Team:Duke/Notebook/July

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Latest revision as of 15:11, 16 October 2014

July 1

Objective: make LB+Cm plates

Four sleeves of plates with 4x500mL medium
34mg/mL chloramphenicol

Objective: insert crRNAs into pdCas9 and scaffold

Plate results

Experimental had lots of colonies, but backbone-only control did as well

reasoning: similarity of BsaI overhangs may lead to re-ligation
solution: treat with CIP before ligation

Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold

plasmids used in 6/30/14 transformation
to make sure BsaI is cutting properly
Results: single band in both appears to be cut DNA

CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)

pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP
Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP
2 hours at 37C (longer than standard protocol)

PCR cleanup of pdCas9 and Repeat scaffold CIP treatments

Final concentrations too low to be of use

Particularly negligible in pdCas9
Need to culture and cut more plasmid to try ligation again

Spread plates from pdCas9 and Repeat scaffold frozen stocks

Used tube “1” for repeat scaffold
Plated on fresh LB+Cm plates

Plates still slightly wet, difficult to spread evenly

Objective: make CCEC stock of DH5alpha-ZI strain

Diluted back 5mL culture in morning

1mL into 5mL LB+Spec

note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable

Inoculated 1 mL culture into each of two 250mL flasks

Flasks contain SOC
30C shaker for overnight growing

July 2

Objective: Make CCEC stock of DH5alpha-ZI strain

Made fresh CCMB-80 buffer stock

precipitate formed during pH adjusting and may have filtered out

could be detrimental to final product

Followed iGEM online protocol to make CCEC from Z1 strain

Overnight culture was much more concentrated than recommended

OD >1
Did not have enough SOC to dilute back fully, so diluted back only a bit

Had to vortex to resuspend cells at two stages of procedure
Final OD after procedure ~1.6 for 100mL of cells
Aliquoted 1mL into tubes, froze at -80C

Next steps:

Test competence with useful strains containing Lac, Tet promoters

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold

3 colonies per plate in LB+Cm medium at 37C overnight
Plates were messy--hard to get individual colonies

July 3

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

Miniprep cultures from 7/2

Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL
Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL

Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold

to insert crRNAs
Digest with BsaI in cutsmart buffer
50 uL DNA in 100 uL reactions with 7.5 uL BsaI
3 hours 20 min at 37C

PCR cleanup of BsaI digest

final concentrations (in 30 uL each):

pdCas9: 65.4, 70.5 ng/uL
Repeat-scaffold: 122.3, 114.8 ng/uL

CIP treatment of pdCas9 and Repeat-scaffold BsaI digests

Two digest tubes pooled into one CIP tube for each backbone
60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer
1.5 hours at 37C

PCR cleanup of CIP treatment

final concentrations (in 30 uL):

pdCas9: 32.5 ng/uL
Repeat scaffold 129.9 ng/uL

Annealed oligos of crRNA inserts for ligation

3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each
5uL each oligo heated to ~95C, slowly brought to rt in water bath

Ligation of crRNA inserts into pdCas9 and Repeat-scaffold

6 working tubes + 5 controls

pdCas9 + each crRNA (GFP1, GFP2, or GFP3)
Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)
pdCas9 Backbone only control
Repeat-scaffold Backbone only control
Insert only controls for each insert

100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned
100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned
1uL annealed oligo insert
10 uL reactions with 0.5 uL T4 Ligase
Transformed into DH5alpha and plated on LB+Cm

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

CCEC Transformation efficiency test

Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC

Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng

Also for use in testing pLac regulation in Z1 strain

Transformed iGEM kit plate 3-6G into DH5alpha-Z1

contains pSB1C3-BBa_I13521: pTet-RBS-mRFP
in order to test regulation of pTet in Z1 strain

July 4

Objective: ligate crRNAs into pdCas9 and Repeat-scaffold

Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:

~1000 colonies on all experimental plates
BO control for pdCas9 has ~500 colonies
BO control for Repeat scaffold has ~1000 colonies
IO controls all have 0 colonies

Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone

12 tubes total in LB+Cm
None from Repeat-scaffold attempts

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

CCEC Test plate results:

130 ng: 100s of colonies
13 ng:~100 colonies
1.3 ng: ~20 colonies
0.13 ng: 0 colonies
0.013 ng: 0 colonies
Competency ~10 colonies/ng (low but working)

Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1

Plate had ~5 colonies

Cultured colonies for Tet and Lac testing in DH5alpha-Z1

One colony of pSB1C3-K741002
One colony of pSB1C3-BBa_I13521
In LB+Cm

July 5

Objective: Insert crRNAs into pdCas9 backbone

Saved stocks of 12 strains in 15% glycerol at -80C

4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9

Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts

4 tubes each
Eluted in 50 uL EB, all concentrations 100-200 ng/uL

Objective: Test effectiveness of DH5alpha-Z1 strain

Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)

Culture of I13521 did not grow

Cultured another colony of pSB1C3-BBa_I13521

from 7/3 plate

July 6

Objective: Test effectiveness of DH5alpha-Z1 strain

Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)

July 7

Objective: insert crRNAs GFP1,2,3 into pdCas9

Analytical restriction digest

12 tubes with potential inserts (4 per crRNA)
pdCas9 as a control
BsaI and XbaI in cutsmart for 1.5 hrs

Agarose gel of restriction digest

Gel 1:

1-4: pdCas9-crRNA-GFP1
5-8: pdCas9-crRNA-GFP2
9-12: pdCas9-crRNA-GFP3

Gel 2:

pdCas9 (control)

Expected single band ~9.3 kb if BsaI cut and ligation worked
Control and uncut failures expect two bands, 5.0 and 4.2 kb
Results: all 12 samples have single band

No uncut plasmids
Could still be recircularized with no insert

gel pictures:

Anneal oligos of 3 crRNA-GFP inserts

Following Dewran’s crRNA protocol
2 uL each oligo diluted in PBS to 20 uL
Placed in 98C water bath, cooled gradually to rt

Phosphorylate oligo inserts of crRNAs

5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK
30 mins at 37C, then deactivated for 20 mins at 65C
Diluted 1/50

Ligation

75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9

Both BsaI cut and CIP treated 7/3/14

1.5 uL diluted, phosphorylated, annealed oligo insert
6 experimental tubes + 2 BO controls and 3 IO controls
Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm

Cultured 2 colonies from pdCas9 plate

For Charlie to try his hands at ligation

Objective: Prepare various backbones for multiple-plasmid use

Transformed two distribution kit parts

Plate 4-6H: pSB4K5-J04450

backbone contains pSC101 origin with Kan resistance

Plate 4-18A: pSB3K3-I20260

backbone contains p15A origin with Kan resistance
On LB+G418 plates

Streaked plate from frozen stock

pSB6A1-J04450

backbone contains pMB1 origin with Amp resistance
on LB+Amp

Objective: Test pTet and pLac in DH5alphaZ1 strain

Streaked plates from frozen stock

pSB1C3-K741002 in DH5alpha-Z1

contains pLac-GFP

pSB1C3-I13521 in DH5alpha-ZI

contains pTet-RFP

On LB+Cm plates

July 8

Objective: insert crRNAs into pdCas9

Plate results from 7/7 transformation

pdCas9-crRNAs: ~50 colonies each
pdCas9 BO: 100s of colonies
Repeat-crRNAs: 100s of colonies
Repeat BO: ~1000 colonies
IO controls: 0 colonies, ~15 for crRNA-GFP3
Notes: unsure why BO controls have more colonies than experimental

Restriction digest of 12 samples from 7/5 prep + pdCas9 control

With SacI/NheI

Should show difference between insert and recircularized backbone

Made and ran 1.5% agarose gel in TBE

for better resolution of small band differences in pdCas9 with/without insert

1: pdCas9 (control)
2-5: pdCas9-crRNA-GFP1 (1-4)
6-9: pdCas9-crRNA-GFP2 (1-4)
10-13: pdCas9-crRNA-GFP3 (1-4)
14: pdCas9 (control)

expected:

3.1, 2.8, and 2.7 kb bands
585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)

Results: One sample, crRNA-GFP3-3, has larger band matching control

This colony may be correct, should be sequenced
Others have smaller band, are probably incorrect

Culture 40 colonies from existing plates to screen for crRNA inserts

10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14
10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14
in LB+Cm

Objective: Prepare various backbones for multiple-plasmid use

Prepared LB+Kan medium

50 ug/mL final concentration Kanamycin

Cultured colonies from transformations and stock streaking

3 colonies each from 3 plasmids:

pSB6A1-J04450 in LB+Amp
pSB3K3-I20260 in LB+Kan
pSB4K5-J04450 in LB+Kan

Objective: Test pLac and pTet in DH5alpha-Z1

Notes:

Need to obtain aTc for pTet testing

Tried to use Doxycycline, but had trouble with solubility in ethanol (?)

pLac is not the final promoter to be used, a different variant will be used

with no glucose dependency

Flow testing of pLac and pTet was suspended until these changes are made

July 9

Objective: Insert crRNAs into pdCas9

Miniprep 40 cultures of potential crRNA inserts in pdCas9

Labeled with three numbers

Date of transformation - crRNA-GFPx - sample number

700uL of each culture saved to freeze in glycerol if colonies appear successful

Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

All 40 tubes + 2 pdCas9 tubes as control
1 uL DNA in 10 uL digest for 2 hrs at 37C
SacI-HF and NheI-HF in cutsmart

Made and ran 1.5% agarose/TBE gels

To differentiate between small band differences
Gel 1:

1. 2-log ladder
2. pdCas9
3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
23. pdCas9
24. 2-log ladder

Gel 2:

1. 2-log ladder
2. pdCas9
3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
23. pdCas9
24. 2-log ladder

Expected results:

pdCas9 and successful inserts:

3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band

unsuccessful recircularizations:

3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band

Results:

Gel 1 appears to only be recircularized
Gel 2 has some promising inserts:

GFP1 samples 1,2, and 3 from 7/7 transformation
GFP2 samples 7,8, and 9 from 7/7 transformation

Glycerol stocks frozen for these six samples
Next Steps:

Run digest with BsaI to confirm that plasmids are not uncut pdCas9
Sequence to confirm presence of insert (using oligos ordered 7/8/14)

Objective: Obtain new backbones and switch inserts into new backbones

Miniprep 3 new backbones

pSB6A1-J04450 (3 copies)

concentrations 437.6, 483.9, and 450.1 ng/uL

pSB3K3-I20260 (3 copies)

concentrations 114.6, 120.0, and 97.3 ng/uL

pSB4K5-J04450 (3 copies)

concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

50 uL DNA in 60 uL total reaction
EcoRI-HF/Spe-HF in cutsmart
3 hours at 37C

Agarose gel extraction of 3 backbones

Gel 1, Left: pSB6A1
Gel 1, Right: pSB3K3
Gel 2, Left: pSB4K5
All 3 lanes had 2 bands as expected

upper band (backbone) in 3K3 had messy trail behind it (not extracted)

All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

pSB1C3-K608012
In order to switch into pSB6A1

Objective: Remake new version of pDGC3

Transformation from iGEM distribution kit plates

Plate 2-6D: pSB1C3-BBa_R0011

Engineered Lac promoter (not affected by glucose)

Plate 3-15O: pSB1C3-BBa_I13500

Strong RBS-GFP

Plated on LB+Cm

July 10

Objective: Obtain backbones and switch parts into new backbones

Gel cleanup of 3 backbones with Zymoclean prep kit

Some tubes had to be divided into two samples for cleanup
Final elution was 20 uL each into 2-3 tubes per backbone
Nanodrops looked unclean: Most had high peak around 220-240 nm

pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL

Peak ~220, then hump ~260

pSB6A1: 270 mg gel = 220 ng/uL

Peak, then hump as before

pSB3K3: 320 mg gel = 53.9 ng/uL

No clear sign of DNA in graph

pSB3K3: 230 mg gel = 20 ng/uL

No DNA

pSB3K3: 130 mg gel = 72 ng/uL

Possibly DNA, best option but may not be useful

pSB4K5: 290 mg gel = 43.4 ng/uL

No clear sign of DNA in graph

pSB4K5: 320 mg gel = 117.3 ng/uL

Possibly DNA, best option but may not be useful

Culture 3 colonies of pSB1C3-K608012

In order to switch into pSB6A1

Objective: Insert crRNAs into pdCas9

Analytical Digest of promising crRNA inserts

XbaI/BsaI digest
In order to confirm that plasmids are not uncut pdCas9
Samples 7-1-1,2,3 and 7-2-7,8,9 (6 tubes) plus pdCas9 as control

Agarose gel of digest results:

1. pdCas9
2-4. pdCas9-crRNA-GFP1, samples 1,2,3
5-7. pdCas9-crRNA-GFP2, samples 7,8,9
Expected:

2 bands at 4.2 and 5.1 kb in pdCas9
1 band at 9.3 kb in successful plasmids with inserts

Results:

pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band
Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.

Objective: Make pDGC3

Culture 3 colonies each from pSB1C3-R0011 and pSB1C3-I13500

Objective: Test effectiveness of DH5alpha-Z1 strain

Mitch and Sargis

Culture pSB1C3-I13521 (pTet-RFP)

For flow
In aTc and non-aTc

July 11

Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of pSB1C3-K608012

concentrations 361, 324, 362 ng/uL

Prep-scale digest of pSB1C3-K608012

2 miniprep tubes, 50 uL DNA in 100 uL each
With EcoRI and SpeI
2+ hrs at 37C

Gel extraction and cleanup to isolate K608012

Cut lower band
Zymoclean prep kit
Each of 2 bands divided into two tubes during cleanup

1A: 280 mg =
1B: 240 mg =
2A: 200 mg =
2B: 200 mg = 62.5 ng/uL (20 uL)

Graphs on nanodrop did not look ideal: peak lower than usual

Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Miniprep pSB1C3-R0011 and pSB1C3-I13500

3 copies each
Concentrations

pSB1C3-R0011: 181, 229, 194 ng/uL
pSB1C3-I13500: 321.5, 228, 300.4 ng/uL

Prep scale digests of two tubes from each miniprep

pSB1C3-R0011 with SpeI/PstI

no extraction necessary

pSB1C3-I13521 with XbaI/PstI

to extract I13521 insert

100 uL total reaction per tube
2-3 hrs at 37C

Agarose gel of pSB1C3-I13500 XbaI/PstI digests

Intended for gel extraction
Expected 2 bands, but only one present
No extraction performed

Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests

Single band appears correct
see below for gel picture

PCR cleanup of pSB1C3-R0011 SpeI/PstI digests

concentrations 124.8, 157.3 ng/uL (30 uL each)

Objective: Switch dCas9-tracrRNA into pSB1C3

Miniprep pdCas9

concentration 167.4 ng/uL

Prep-scale digest of pSB1C3-R0011

1 miniprep with XbaI/PstI
To extract pSB1C3 backbone

Agarose gel of pSB1C3-R0011 XbaI/PstI digest

Intended for gel extraction
Expected 2 bands, but only one present
No extraction performed

PCR of dCas9-tracrRNA and anti-tracrRNA

4 tubes each, with pdCas9 as template in 0, 0.4, 0.7, and 1.0 uL quantities
oligos dCas9tracr-up and dCas9tracr-dn for dCas9-tracrRNA PCR
oligos dCas9tracr-up and tracrRNA-dn for anti-tracrRNA PCR
Q5 polymerase, with 64C annealing and 2 min extension

Agarose gel of PCR (and digest) results:

Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests
Results:

dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes
tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension.

tracrRNA PCR should be done with shorter extension time

PCR cleanup of dCas9-tracrRNA PCR

Concentration >200 ng/uL

Objective: Test BsaI and XbaI enzymes

Analytical digest of dCas9

One tube with BsaI/EcoRI
One tube with XbaI/EcoRI

XbaI from tube with exp. 5/14

One tube with XbaI/EcoRI

XbaI from tube with exp. 2/15

Agarose gel of digest:

1. BsaI/EcoRI: expected 3 bands at 2.7, 2.9, and 3.5 kb
2. XbaI (5/14) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
3. XbaI (2/15) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
Results: Lanes 1 and 3 look as expected. Lane two has one extra band at 3.5 kb corresponding to partial digestion with XbaI. 5/14 XbaI tube is ineffective and has been thrown out

July 14

Objective: insert crRNAs into pdCas9

Sequencing Order #1608

BigDye reaction with BD buffer 1.1

pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9up1

pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9up1

pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9up1

pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9up1

pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9up1

pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9up1

pdCas9-crRNA_GFP3 sample 3 w/ pdCas9up1

pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9dn1

pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9dn1

pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9dn1

pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9dn1

pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9dn1

pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9dn1

pdCas9-crRNA_GFP3 sample 3 w/ pdCas9dn1

Results:

pdCas9-crRNA_GFP1 use sample 7-1

pdCas9-crRNA_GFP2 use sample 7-9

pdCas9-crRNA_GFP3 need to find more colonies

Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Prep scale digest of pSB1C3-I13500 with XbaI/PstI

Tube 3 from 7/11/14 miniprep
To extract both I13500 and pSB1C3 backbone
100 uL reaction with 50 uL DNA in cutsmart

Agarose gel extraction and cleanup of pSB1C3 and I13500

Top band (~2kb): pSB1C3
Bottom band (~1kb): I13500
Zymoclean prep kit
Concentrations:

pSB1C3: 319 ng/uL in 20 uL (dirty)
I13500: 91.2 ng/uL in 20 uL (dirty)

Ligation of pSB1C3-R0011 and I13500

100ng = 0.7 uL pSB1C3-R0011 (#2) cut with SpeI/PstI on 7/11
150ng = 1.8 uL I13500 cut with XbaI/PstI on 7/14
Experimental, BO control, and IO control
rt for 1 hr
Heat shock transformation into DH5alpha and plated on LB+Cm

Objective: Switch K608012 into pSB6A1

Ligation of pSB6A1 and K608012

100 ng = 0.5 uL pSB6A1 (“Yellow” sample) cut with EcoRI/SpeI on 7/9
150 ng = 2.5 uL K608012 (#2B) cut with EcoRI/SpeI on 7/11
Experimental, BO control, and IO control
rt for 1 hr
Heat shock transformation into DH5alpha and plated on LB+Amp

Objective: Switch dCas9-tracrRNA into pSB1C3

Prep scale digests

dCas9-tracr PCR with XbaI/PstI (no extraction)
pSB1C3-K608012 (tube 3) with XbaI/PstI (to obtain pSB1C3)
100 uL reaction each with 50 uL DNA in cutsmart

PCR cleanup of dCas9-tracr digest

Qiagen miniprep kit
Concentration 38.1 ng/uL in 30 uL

Agarose gel extraction and cleanup of pSB1C3

gel order: left K608012, right I13500
extracted pSB1C3, top band of K608012 (~2 kb)
Zymoclean kit
Concentration 127.9 ng/uL in 20 uL (dirty)

Ligation of pSB1C3 and dCas9-tracrRNA PCR

100 ng = 3 uL dCas9-tracrRNA PCR cut with XbaI/PstI on 7/14
150 ng = 0.6 uL pSB1C3 (from I13500) cut with XbaI/PstI on 7/14
Experimental, BO control, and IO control
rt for 1 hr
Heat shock transformation into DH5alpha and plated on LB+Cm

Objective: Create pTet-anti-tracrRNA

Prep-scale digest of pSB1C3-R0040

With SpeI/PstI (no extraction necessary)
100 uL reaction with 50 uL DNA in cutsmart
4 hrs at 37C

PCR anti-tracrRNA from pdCas9

Oligos dCas9tracr-up and tracrRNA-dn
0, 0.4, 0.7, and 1.0 uL pdCas9 template in 50 uL reactions
Q5 polymerase
annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR

Results: only 1.0 uL template lane appeared to amplify

Need to make more using this template concentration

PCR anti-tracrRNA from pdCas9

Oligos dCas9tracr-up and tracrRNA-dn
1.0 uL pdCas9 template in each 50 uL reaction
8 tubes with Q5 polymerase
annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR and pSB1C3-R0040 digest

Lanes 1-8: anti-tracrRNA PCR
Lane 9: R0040 digest
Results:

PCR appears to work in all lanes (band size ~0.1 kb)
R0040 digest single band appears correct

PCR cleanup of anti-tracrRNA PCR and pSB1C3-R0040

Concentrations:

anti-tracrRNA: 83.3 ng/uL in 30 uL
pSB1C3-R0040: 25 ng/uL in 30 uL

July 15

Objective: Create pTet-anti-tracrRNA

Digest of anti-tracrRNA PCR

with XbaI, PstI-HF, and DpnI
3 hrs at 37C
60 uL DNA in 100 uL total reaction

PCR Cleanup of anti-tracrRNA PCR digest and pSB1C3-R0040 digest

Concentrations

anti-tracrRNA: 100 ng/uL
pSB1C3-R0040: 35 ng/uL

Ligation of anti-tracrRNA and pSB1C3-R0040

100 ng = 3 uL pSB1C3-R0040 cut with SpeI/PstI
15 ng = 0.15 uL anti-tracrRNA PCR cut with XbaI/PstI/DpnI
Experimental, Backbone only, and Insert only
Transformed via heat shock into DH5alphas and plated on LB+Cm

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

Experimental: 8 colonies
BO Control: 4 colonies
IO Control: 7 colonies

Culture colonies

4 copies of potential pSB1C3-dCas9-tracrRNA construct

Digest of dCas9-tracrRNA PCR and heat inactivation

Digest with DpnI
22 uL DNA in 30 uL reaction
1 hr at 37C, then 30 mins at 80C to heat-inactivate
assumed concentration 20 ng/uL

PCR Cleanup of pSB1C3 digest

2 tubes of gel-extracted and cleaned pSB1C3 cut with XbaI and PstI
11 ug original yields 309.6 ng/uL in 20 uL “superclean”

Ligation of dCas9-tracrRNA PCR and pSB1C3

150 ng = 0.5 uL pSB1C3 cut with XbaI and PstI
100 ng = 5 uL dCas9-tracrRNA PCR cut with Xbal/PstI/DpnI
Experimental, Backbone only, and Insert only
Transformed via heat shock into DH5alphas and plated on LB+Cm

Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

Lawn of colonies on Experimental and BO control
No colonies on IO control

Digest of pSB1C3-R0011

With SpeI, PstI, and CIP
50 uL DNA in 100 uL reaction
2.5 hrs at 37C

PCR Cleanup of pSB1C3-R0011 digest and I13500 digest

pSB1C3-R0011 concentration 175 ng/uL
I13500 (from previous digestion and extraction)

2.3 ug original yields 124 ng/uL in 20 uL “superclean”

Ligation of I13500 and pSB1C3-R0011

100 ng = 0.6 uL pSB1C3-R0011 cut with SpeI/PstI/CIP
150 ng = 1.2 uL I13500 cut with XbaI/PstI
Experimental, Backbone only, and Insert only
Transformed via heat shock into DH5alphas and plated on LB+Cm

Objective: Switch K608012 into pSB6A1

Plate results:

2 colonies on experimental plate
1 colony on BO, no colonies on IO

Cultured colonies

2 colonies of potential pSB6A1-K608012 construct

PCR Cleanup of pSB6A1 digest and K608012 digest

pSB6A1, 2 tubes gel extracted and cleaned

10 ug original yields 203 ng/uL in 30 uL

K608012, 3 tubes gel extracted and cleaned

4 ug original yields 73.0 ng/uL in 30 uL

Ligation of K608012 and pSB6A1

100 ng = 0.5 uL pSB6A1 cut with EcoRI and SpeI
150 ng = 2 uL K608012 cut with EcoRI and SpeI
Experimental, Backbone only, and Insert only
Transformed via heat shock into DH5alphas and plated on LB+Amp

July 16

Objective: Create pTet-anti-tracrRNA

Plate results:

Experimental: over 1000 colonies
BO control had ~500 colonies, IO control had ~50 colonies

Cultured colonies

4 copies of potential pSB1C3-R0040-anti-tracrRNA

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

Experimental: 12-15 colonies
BO control had 3 colonies, IO control had ~30 colonies

Minipreps

4 copies of potential pSB1C3-dCas9-tracrRNA construct
From 7/14 ligation
concentrations: 537.2, 45.4, 79.1, and 71.5 ng/uL

Only one has normal concentration, others very low

Analytical digest of pSB1C3-dCas9-tracrRNA constructs:

4 copies with NheI/XbaI

Agarose gel of digests:

Lanes 1-2: pSB6A1-K608012 with MfeI/XbaI (expected 4.1, 0.6 kb bands)
Lanes 3-6: pSB1C3-dCas9-tracrRNA with NheI/XbaI (expected 5.0, 1.5 kb bands)
Results: gel was fuzzy and unclear

lanes 2,3,5,6 clearly incorrect: 1 band ~2.0 kb
lane 1 had one fuzzy band around 4-5 kb (could be incomplete digest)
lane 4 had band around 5 kb with smear above (could be incomplete digest)

New analytical digest of pSB1C3-dCas9-tracrRNA construct

Only copy #2 (showed potential in previous digest
With KpnI/SpeI for 2 hours at 37C
Also put previous digest back in bath for 2 more hours

Agarose gel of digests

DETAILS

Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

~500 colonies on experimental plate
BO control had over 1000 colonies
IO control had no colonies

Cultured colonies

4 copies of potential pSB1C3-R0011-I13500 construct

Objective: Switch K608012 into pSB6A1

Plate results:

0 colonies on experimental plate
4 colonies on BO control, 1 colony on IO control

Objective: Insert crRNAs into pdCas9

Colony PCR of pdCas9-GFP3 plate

Plate from 7/7/14
15 colonies plus pdCas9 miniprep as control
double dipped in 50 uL Taq poly mix and 50 uL SOC
Oligos pdCas9-up1 and pdCas9-dn1
TALCOLONYPCR protocol with 64C anneal

Agarose gel of colony PCR

Expected to see higher band matching control in successful colonies
lanes 1 and 17 are pdCas9 control
assay looks helpful, successes in samples 3,5,6,8,9,12,13,15

Cultured colonies

Copies 3,5,6, and 8 from colony PCR in LB+Cm

July 17

Objective: Create pTet-anti-tracrRNA and pSB1C3-R0011-I13500

Minipreps

4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation

4 copies of pSB1C3-R0011-I13500 from 7/15 ligation

Analytical digest of minipreps

pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI
pSB1C3-R0011-I13500 with MfeI/NdeI/SacI

Agarose gel of digest results:

Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, <0.1 kb bands)
Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)
Results:

R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly
R0011-I13500 does not look correct (only one band at 2.0 kb)

Objective: Switch dCas9-tracrRNA into pSB1C3

Colony PCR on pSB1C3-dCas9-tracrRNA plate

Ligation plate from 7/15/14
15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC
oligos SB1C3-up1 and SB1C3-dn1
annealed at 65C, extension 2 mins

Agarose gel of colony PCR

results: primer-bands only (no successes)

Objective: Switch K608012 into pSB6A1

Culture colonies

2 copies of pSB6A1-J04450 (from 6-9-14 plate)
2 copies of pSB1C3-K608012

July 18

Objective: Switch dCas9-tracrRNA into pSB1C3

PCR of dCas9-tracrRNA from pdCas9

8 tubes, each with 1 uL pdCas9 template at 1 ng/uL
oligos dCas9tracr-up and dCas9-tracr-dn
2 min extension, 64C anneal

Agarose gel of PCR product

All 8 tubes look successful (band ~4 kb)

PCR cleanup of dCas9-tracrRNA

Qiagen kit
Into 2 tubes, 30 uL each

Prep-scale digest of PCR product

with PstI/XbaI/DpnI
in 100 uL each for 3 hrs at 37C

Treat pSB1C3 digest with CIP

1 hr at 37C

Objective: Switch K608012 into pSB6A1

Miniprep pSB1C3-K608012 and pSB6A1-J04450

1 copy each (the second copy of each did not grow in culture)
concentrations:

pSB1C3-K608012: 344 ng/uL
pSB6A1-J04450: 421.5 ng/uL

Prep-scale digest of K608012 and pSB6A1

50 uL DNA in 100 uL reaction
Both with EcoRI and PstI
3 hrs at 37C

CIP treatment of pSB6A1 and heat-inactivation

2.5 uL CIP added to digest tube
Both digests returned to 37C for 1 hr
Then 30 mins at 80C to heat-inactivate

Gel extraction of pSB6A1 and K608012

Gel had no EtBr added, and was discarded (product was lost)

July 20

Objective: culture colonies for various purposes

Cultured colonies

4 copies pSB6A1-J04450

2 from older plate, 2 from newer

4 copies pSB1C3-K608012

2 from older plate, 2 from newer

4 copies pSB1C3-B0030

To obtain pSB1C3 without gel extraction

4 copies pSB1C3-R0011
4 copies pSB1C3-I13500

July 21

Objective: Sequence to confirm constructs

Sequencing reaction and Order #1634

1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1
5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1
9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1
11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1
Big Dye, buffer version 1.1

Order 1634 Results:

pdCas9-GFP3 copies 3, 6, and 8 are correct

copy 5 has a single mutation

anti-tracrRNA: both copies are correct

copy 1 was a cleaner, more reliable

Objective: Create pSB1C3-R0011-I13500

Minipreps

Qiagen kit
4 tubes of pSB1C3-R0011

concentrations 255.7, 169.2, 204.8, 151.2 ng/uL

4 tubes of pSB1C3-I13500

concentrations 148.9, 214.2, 260.7, 432.5

Prep scale digests of pSB1C3-R0011 and I13500

pSB1C3-R0011 with SpeI/PstI, samples 1,3
pSB1C3-I13500 with XbaI/PstI, samples 1,3
50 uL DNA in 100 uL each reaction, 2 tubes per construct
3 hrs at 37C
Added CIP (2.5 uL) to pSB1C3-R0011 reaction
1 more hr for both at 37C
Heat inactivation of both reactions: 30 mins at 80C

Gel extraction of I13500

extraction gel 1, lanes 1 and 2
Zymoclean prep kit
sample 1 (lane 1): 300 mg gel yields
sample 3 (lane 2): 550 mg gel yields

PCR cleanup of pSB1C3-R0011

Qiagen kit
Two tubes of digest combined into one tube product
Concentration

Ligation of pSB1C3-R0011 and I13500

Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL
New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL
Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL
New insert (I13500 extracted 7/21): 300 ng = 3 uL
Ligations numbered 1-8:

1. OB+OI
2. OB+NI
3. NB+OI
4. NB+NI
5. OB control
6. OI control
7. NB control
8. NI control

1 hr at rt
Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch dCas9-tracrRNA into pSB1C3

Minipreps

Qiagen kit
4 tubes of pSB1C3-B0030

to obtain pSB1C3 without gel extraction
concentrations 116.8, 99.9, 115.3, 145.1 ng/uL

PCR cleanup of dCas9-tracrRNA PCR

Product digested 7/18 with XbaI/PstI/DpnI
Concentration 80.5 ng/uL in 30 uL
Heat inactivation of cleaned product, 30 mins at 80C

Prep scale digest of pSB1C3-B0030

With XbaI/PstI, samples 1,3
50 uL DNA in 100 uL each reaction, 2 tubes
3 hrs at 37C
Added CIP (2.5 uL)
1 more hr for both at 37C
Heat inactivation: 30 mins at 80C

PCR cleanup of pSB1C3

B0030 should not be retained
Qiagen kit
Two tubes of digest combined into one tube product
Concentration

Ligation of pSB1C3 and dCas9-tracrRNA

Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL
Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL
New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL
Ligations numbered 9-13:

9. B+OI
10. B+NI
11. B control
12. OI control
13. NI control

1 hr at rt
Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch K608012 into pSB6A1

Minipreps

Qiagen kit
4 tubes of pSB6A1-J04450

concentrations 189.3, 432.5, 688.7, 487.3 ng/uL

4 tubes of pSB1C3-K608012

concentrations 68.8, 91.6, 346.1, ng/uL

Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012

pSB6A1-J04450 with XbaI/SpeI, samples 1,3
pSB1C3-K608012 with EcoRI/PstI, samples 1,3
50 uL DNA in 100 uL each reaction, 2 tubes per construct
3 hrs at 37C
Added CIP (2.5 uL) to pSB6A1-J04450 reaction
1 more hr for both at 37C
Heat inactivation of both reactions: 30 mins at 80C

Gel extraction of pSB6A1 and K608012

K608012: gel 1, lane 3 and gel 2, lane 1
pSB6A1: gel 2, lanes 2 and 3
Zymoclean prep kit
K608012 sample 1 (lane 1-3): 300 mg gel yields
K608012 sample 3 (lane 2-1): 550 mg gel yields
pSB6A1 sample 1 (lane 2-2):
pSB6A1 sample 3 (lane 2-3):

Ligation of pSB6A1 and K608012

Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL
Old insert (K608012 extracted 7/15): 300 ng = 4 uL
Ligations numbered 14-16:

14. B+I
15. B control
16. I control

1 hr at rt
Transformed via heat shock into DH5alphas, plated on LB+Amp

Gibson of pSB6A1 and K608012

New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
Numbered 17-19:

17. B+I
18. B control
19. I control

15 uL Mert’s DIY Gibson mix
1 hr at 60C
Transformed via heat shock into DH5alphas, plated on LB+Amp

SLIC of pSB6A1 and K608012

New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
Numbered 20-22:

20. B+I
21. B control
22. I control

2.5 mins at rt, then 10 mins at 4C
Transformed via heat shock into DH5alphas, plated on LB+Amp

July 22

Objective: Create pSB1C3-R0011-I13500

Plate results:

All four experimental plates had lots of colonies
both Backbone-only controls had similar colony numbers to experimentals
Insert-only controls had no colonies

Colony PCR

4 copies each of 4 experimental plates = 16 colonies
Oligos SB1C3-up and SB1C3-dn with Taq polymerase
66C anneal temp, 2 min extension time

Agarose gel of colony PCR results

1-4: OB+OI pSB1C3-R0011-I13500
5-8: OB+NI pSB1C3-R0011-I13500
9-12: NB+OI pSB1C3-R0011-I13500
13-16: NB+NI pSB1C3-R0011-I13500
17-20: B+OI pSB1C3-dCas9-tracrRNA
21-24: B+NI pSB1C3-dCas9-tracrRNA

Results: no successful bands on any colonies

Just primer-dimers or small amplified bands?

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

Both experimental plates had lots of colonies
Backbone-only controls had similar colony numbers to experimentals
Insert-only controls had no colonies

Colony PCR

4 copies each of 2 experimental plates = 8 colonies
Oligos SB1C3-up and SB1C3-dn with Taq polymerase
66C anneal temp, 2 min extension time

Agarose gel of colony PCR results

see above for gel order

Results: no successful bands on any colonies

Just primer-dimers or small amplified bands?

Objective: Switch K608012 into pSB6A1

Plate results:

No colonies on Gibson or SLIC plates
3 colonies on Ligation plate
A few colonies on controls, but not many

Cultured colonies

3 colonies of potential pSB6A1-K608012 from Ligation plate
in LB+Amp

Ligation of K608012 and pSB6A1

Same constructs as 7/21 ligation
This time let recover in SOC for 2 hrs instead of 1 hr
Plated on LB+Amp

Objective: Try putting K608012 into pSB1A2 instead

Transformation from kit plate

Plate 4-1N

pSB1A2-B0034

Plated on LB+Amp

July 23

Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of potential pSB6A1-K608012

Qiagen kit
Saved stocks in 15% glycerol

Analytical digest of minipreps

With NdeI in Cutsmart, 10 uL final volume

Agarose gel of analytical digest

Results:

Copies 1 and 2 are strange and incorrect
Copy 3 looks correct
Need to check with more digests

Analytical digest to check promising sample

Copy 3 in 3 different reactions

EcoRI/SpeI
EcoRI/MfeI
NcoI/PvuI

Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI
Control: pSB1C3-K608012 with MfeI/NcoI

Agarose gel of analytical digest

1. Ligation 3 with EcoRI/SpeI
2. Ligation 3 with EcoRI/MfeI
3. Ligation 3 with NcoI/PvuI
4. pSB6A1-J04450 with EcoRI/SpeI/PvuI
5. pSB1C3-K608012 with MfeI/NcoI

Results:

Sample looks good
Incorrect samples 1 and 2 glycerol stocks thrown out

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Treat pSB4K5 with CIP

1.25 uL CIP in 25 uL reaction
pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9

Prep-scale digest to obtain R0040-anti-tracrRNA

pSB1C3-R0040-anti-tracrRNA
with EcoRI/SpeI

Gel extraction and cleanup of R0040-anti-tracrRNA

Extracted smaller band
Attempted to clean up using Qiagen prep kit

dissolved with Zymoclean ADB
Then followed Qiagen PCR cleanup protocol
510 mg gel yielded little or no DNA

Objective: Create pSB1C3-R0011-I13500

Colony PCR

8 colonies from 7/21 ligation plates
Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011

Agarose gel of colony PCR results

No successes
Controls did not work properly either
colony PCR does not seem to be effective--need to just pick colonies

Culture colonies

8 colonies of potential pSB1C3-R0011-I13500
From 7/21 ligation plates (colonies from today’s colony PCR)

Objective: Measurement interlab study

Transformations from kit plates

Plate 1-20K

pSB1C3-K823005

Plate 1-22I

pSB1C3-K823012

Plate 2-24B

pSB1C3-E0240

July 24

Objective: Create pSB1C3-R0011-I13500

Miniprep 8 colonies of potential pSB1C3-R0011-I13500

Qiagen kit

Analytical digest of minipreps

8 copies with NcoI
8 copies with EcoRI/SpeI

Agarose gel of digest results

1-8: copies 1-8 with NcoI
9-16: copies 1-8 with EcoRI/SpeI
Results: no successes, insert appears to be missing

Unsure whether incorrect colonies are pSB1C3 or pSB1C3-R0011

Agarose gel of digest results

Thicker gel, longer run time, more DNA
from same digest tubes
Results:

no small band on EcoRI/SpeI cut and no difference in larger band for the two cuts, indicating that R0011 is not present

Ligation of pSB1C3-R0011 and I13500

Used lab stock of T4 Ligase and buffer rather than iGEM stock
100 ng pSB1C3-R0011 = 2.5 uL
300 ng I13500 = 3 uL
4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
BO and IO controls for each time
Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Switch dCas9-tracrRNA into pSB1C3

Ligation of pSB1C3 and dCas9-tracrRNA

Used lab stock of T4 Ligase and buffer rather than iGEM stock
300 ng pSB1C3 = 3 uL
100 ng dCas9-tracrRNA PCR = 1 uL
4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
BO and IO controls for each time
Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Test GFP repression by crRNAs

Transform pSB6A1-K608012 into DH5alpha-ZI

Plated on LB+Amp

Objective: Obtain constructs for various uses

Streaked plate from frozen stock

One LB+Cm plate in thirds:

pSB1C3-R0040-antitracrRNA
pSB1C3-Repeat-seq scaffold
pSB1C3-Csy4-gRNA scaffold

Objective: Measurement interlab study

Cultured colonies (2 each)

pSB1C3-E0240
pSB1C3-K823005
pSB1C3-K823012
pSB3K3-I20260

July 25

Objective: Create pSB1C3-R0011-I13500

Plate results:

no apparent difference between 15, 30, 45, or 60 minute ligations
many colonies on all Experimental and BO control plates
no colonies on IO control

Colony PCR:

16 colonies from pSB1C3-R0011-I13500 plates

4 from each ligation time

Taq polymerase with oligos SB1C3-up and SB1C3-dn
66C anneal temp, 45 sec extension

Agarose gel of colony PCR

No successes
It seems as though colony PCRs are not working--try control with a gradient

Gradient Colony PCR

to optimize with SB1C3-up and SB1C3-dn primers
template colonies of pSB1C3-K608011 from plate
anneal temps 62-72 C, extension time 45 sec

Agarose gel of gradient colony PCR

left to right: 62C to 72C anneal temps
Results: 62C was the best anneal temp--worked well

gradual decrease as temp increased, no amplification above 65C

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

no apparent difference between 15, 30, 45, or 60 minute ligations
many colonies on all Experimental and BO control plates
no colonies on IO control

Colony PCR:

16 colonies from pSB1C3-dCas9-tracrRNA plates

4 from each ligation time

Taq polymerase with oligos SB1C3-up and SB1C3-dn
66C anneal temp, 45 sec extension

Agarose gel of colony PCR

No successes
It seems as though colony PCRs are not working--try control with a gradient

see gradient results above

Objective: Various ligations

Prep-scale digests

pSB1C3-Csy4-scaffold (2 copies) with XbaI/PstI

to ligate into R0040

pSB1C3-Repeat-seq scaffold (2 copies) with XbaI/PstI

to ligate into R0040

pSB1C3-R0040-anti-tracrRNA (2 copies) with XbaI/PstI
100 uL reactions each tube

PCR cleanups on all digests

Qiagen kit
Concentrations (in 50 uL each):

pSB1C3-Csy4-scaffold: 55.9, 105.5 ng/uL
pSB1C3-Repeat-seq scaffold: 15.2, 55.2 ng/uL
pSB1C3-R0040-anti-tracrRNA: 90.6 ng/uL

Need to extract from gels before using, only cleaned up for safer storage

Objective: Measurement interlab study

Minipreps

Qiagen kit

pSB1C3-K823005 (2 copies)

concentrations 256.3, 314.8 ng/uL

pSB1C3-K823012 (2 copies)

concentrations 204.4, 192.5 ng/uL

pSB1C3-E0240 (2 copies)

concentrations 266.3, 241.5 ng/uL

Prep-scale digests

pSB1C3-K823005 (2 copies) with SpeI/PstI
pSB1C3-K823012 (2 copies) with SpeI/PstI
pSB1C3-E0240 (2 copies) with XbaI/PstI
100 uL each tube

PCR cleanups on all digests

Qiagen kit
Concentrations (in 50 uL each):

pSB1C3-K823005: 156.3, 181.7 ng/uL
pSB1C3-K823012: 110.0, 98.0 ng/uL
pSB1C3-E0240: 110.0, 114.6 ng/uL

Objective: Test crRNA repression of GFP reporter

Cultured colony

To make CCEC of reporter strain
One colony of pSB6A1-K608012 in DH5alpha-ZI
Grew for 8 hrs during the day in 2 mL LB+Amp at 37C
Inoculated into 100 mL flask of LB+Amp for overnight shaker at rt

Flow cytometry of reporter strain

quick check to make sure reporter still works with new backbone
Used parameters from previous GFP flow data
Levels slightly under what was seen before, but still strong

July 26

Objective: Test crRNA repression of GFP reporter

Prepared Chemically competent E. coli

Using DH5alpha-ZI + pSB6A1-K608012
iGEM CCEC protocol with smaller volumes (made ~20 1-mL aliquots)

Double-Transformation of pdCas9 variants into reporter cells

Charlie Cooper

pdCas9 (with BsaI insert as crRNA sequence)
pdCas9-crRNA_GFP1
pdCas9-crRNA_GFP2
pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Colony PCRs

16 colonies from 7/24 ligation plates
oligos SB1C3-up and SB1C3-dn
62C anneal (as determined by gradient), 45 sec extension

Agarose gel of Colony PCRs

Results: colonies 5 and 14 amplified at the correct size

Culture colonies

Colonies 5 and 14 from colony PCR of potential pSB1C3-R0011-I13500

Objective: Create pSB1C3-dCas9-tracrRNA

Colony PCRs

16 colonies from 7/24 ligation plates
oligos SB1C3-up and SB1C3-dn
62C anneal (as determined by gradient), 45 sec extension
PCRs left in machine without starting reaction for ~1 hr, could have kill reaction

Agarose gel of Colony PCRs

No successes

July 27

Objective: Test crRNA repression of GFP reporter

Cultured colonies for flow

3 copies of each
DH5alpha-Z1 (background)
DH5-alpha-Z1 + pSB6A1-K608012 (reporter positive control)
DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9 (non-target control)
DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP1
DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP2
DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Minipreps

Colonies 5 and 14 from 7/26 colony PCRs
Qiagen kit

July 28

Objective: Create pSB1C3-R0011-I13500

Analytical digest of minipreps

Colonies 5 and 14 of potential pSB1C3-R0011-I13500
with MfeI
with NcoI
with EcoRI/PstI

Agarose gel of analytical digest results

Gel Order:

#14 w/ MfeI
#14 w/ EcoRI/PstI
#14 w/ NcoI
#5 w/ MfeI
#5 w/ EcoRI/PstI
#5 w/ NcoI

Results: All looks correct, except that MfeI cut once instead of twice

could be pSB1C3-I13500 without R0011

Objective: Insert mCherry G-block into pSB1C3-R0011-I13500

PCR of mCherry G-block

Q5 polymerase with Oligos SB1C3-dn and pDGC3-up
4 tubes with 0, 0.4, 0.7, 1.0 uL template at 1 ng/uL
45 sec extension, 66C anneal temp

Agarose gel of PCR

3 tubes look successful

PCR cleanup of mCherry G-block PCR

Qiagen kit
40 uL at 27.6 ng/uL

Objective: Various ligations

Gel extraction and cleanup

Each extraction included 2 cleaned digests combined into one lane (~100 uL)
Cut and cleaned with Zymoclean prep kit
pSB1C3-Csy4-scaffold cut with XbaI/PstI on 7/25

pSB1C3 backbone 47.5 ng/uL in 30 uL
Csy4-scaffold insert 15.4 ng/uL in 30 uL

pSB1C3-Repeat-seq scaffold cut with XbaI/PstI on 7/25

pSB1C3 backbone 74.8 ng/uL in 30 uL
Repeat-seq scaffold insert 13.1 ng/uL in 30 uL

pSB1C3-R0040-anti-tracrRNA cut with XbaI/PstI on 7/25

pSB1C3 backbone 164.0 ng/uL in 30 uL
R0040-anti-tracrRNA 176.0 ng/uL in 30 uL

PCR Cleanup on gel extraction cleanups

to “superclean”
pSB1C3 4 tubes combined into 1: 469.6 ng/uL in 20 uL
Repeat-seq scaffold: 23.9 ng/uL

rough graph, probably not useful

Csy4 scaffold: 16 ng/uL

rough graph, probably not useful

R0040-anti-tracrRNA: 7.4 ng/uL

rough graph, probably not useful

Objective: Measurement interlab study

CIP treatment of backbones

Using Charlie’s CIP stock
pSB1C3-K823005 cut with SpeI/PstI on 7/25 (2 tubes)
pSB1C3-K823012 cut with SpeI/PstI on 7/25 (2 tubes)

Gel extraction and cleanup

2 cleaned digests combined into one lane (~100 uL)
Cut and cleaned with Zymoclean prep kit
pSB1C3-E0240 cut with XbaI/PstI on 7/25

pSB1C3 backbone 116.4 ng/uL in 30 uL
E0240 86.5 ng/uL in 30 uL

PCR Cleanup of CIP treatment and gel extraction cleanup

Qiagen kit
pSB1C3-K823005 SpeI/PstI CIP treated: 119.8, 119.7 ng/uL
pSB1C3-K823012 SpeI/PstI CIP treated: 60.9, 62.3 ng/uL
E0240 XbaI/PstI extracted: 56.5 ng/uL in 20 uL (“superclean”)

Ligations

E0240 into two backbones: pSB1C3-K823005 and pSB1C3-K823012
pSB1C3-K823005 SpeI/PstI/CIP: 100 ng = 0.87 uL
pSB1C3-K823012 SpeI/PstI/CIP: 100 ng = 1.67 uL
E0240 XbaI/PstI: 300 ng = 6 uL
2 BO controls and 1 IO control
20 mins at rt
Heat shock transformation and plated on LB+Cm

Objective: Test crRNA repression of GFP reporter

Dilute cultures to grow in log phase for flow

1/1000 dilution into 50 mL
18 samples: 3 copies each of 6 strains (labelled 2-19)
Samples 2-4: DH5alpha-Z1 (background)

grown in LB+Spec

Samples 5-7: DH5alpha-Z1 + pSB6A1-K608012 (reporter positive control)

grown in LB+Amp

Samples 8-10: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9 (non-target control)

grown in LB+Amp+Cm

Samples 11-13: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP1

grown in LB+Amp+Cm

Samples 14-16: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP2

grown in LB+Amp+Cm

Samples 17-19: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP3

grown in LB+Amp+Cm

Flow cytometry

Samples grown for 4.5 hrs after initial dilution
1/50 final dilution of sample into PBS, placed on ice until run
parameters (same as previous GFP runs):

FSC 350V log3
SSC 350V log3
B1 350V log5
Trigger 10
10,000 event limit (100,000 events used in the past)

Results

~3-fold repression of reporter with crRNA_GFP1 or crRNA_GFP3
crRNA_GFP2 did not repress
non-target control repressed little or not at all
See document “Flow stats 7-28-14” for data details

July 29

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Minipreps

pSB4K5-J04450 (4 copies)
pSB1C3-R0040-anti-tracrRNA (4 copies)

Prep-scale digest

pSB4K5-J04450 (4 copies) with EcoRI/SpeI
pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI

Gel extraction and cleanup of digests

Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450

2 digests per gel well
2.04 g total gel eluted into two tubes:

51.4 ng/uL and 94.5 ng/uL, 30 uL each

Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA

2 digests per gel well
1.61 g total gel eluted into two tubes:

99.4 ng/uL and 82.3 ng/uL, 30 uL each

Objective: Create pSB1C3-R0011-I13500

Colony PCR

24 colonies of potential pSB1C3-R0011-I13500

8 colonies from 7/15 ligation
4x4=16 colonies from 4 different 7/21 ligation plates

Taq polymerase with oligos SB1C3-up and SB1C3-dn
extension time 45 sec, anneal temp 62C

Agarose gel of colony PCR results

colony #19 is only promising result

Analytical digest

pSB1C3-R0011 copies 2, 4
pSB1C3-I13500 copies 2, 4
with MfeI/NcoI
To test whether enzymes or original parts are the source of ligation difficulties

Agarose gel of digest results

Order:

1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)
2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)
3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)
4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)

Results:

R0011 both copies only show one band
I13500 both copies appear correct

Cultured colonies

Potential pSB1C3-R0011-I13500 #19 from colony PCR
pSB1C3-R0011 (3 copies) from original transformation

suspicion that our R0011 is incorrect--could be just bad copies

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

Culture colonies

pSB1C3-R0040 (4 copies)

Objective: Measurement interlab study

Plate results

both experimental and both backbone-only plates all had hundreds of colonies
insert-only controls had no colonies

Colony PCR

8 colonies of potential pSB1C3-K823005-E0240
8 colonies of potential pSB1C3-K823012-E0240
Taq polymerase with oligos SB1C3-up and SB1C3-dn
extension time 45 sec, anneal temp 62C

Agarose gel of colony PCR results

1-8: pSB1C3-K823005-E0240
9-16: pSB1C3-K823012-E0240
Results: no successes

July 30

Objective: Create pSB1C3-R0011-I13500

Minipreps

pSB1C3-R0011-I13500 #19

concentration 246.4 ng/uL

pSB1C3-R0011 (3 copies)

concentrations 187.6, 178.8, 212.8 ng/uL

Analytical digest

pSB1C3-R0011-I13500 #19

three digests in cutsmart, 10 uL final volume each

NcoI
MfeI
XbaI/PstI

pSB1C3-R0011 (3 copies)

with NcoI/MfeI in cutsmart, 10 uL final volume

Agarose gel of digest results

1. pSB1C3-R0011-I13500 #19 with NcoI
2. pSB1C3-R0011-I13500 #19 with MfeI
3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI
4. pSB1C3-R0011 #1 with NcoI/MfeI
5. pSB1C3-R0011 #2 with NcoI/MfeI
6. pSB1C3-R0011 #3 with NcoI/MfeI
Results: hard to explain

R0011-I13500 not correct
R0011 all three copies only cut once

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Treat pSB4K5 with Antarctic Phosphatase

2 copies digested on 7/29, combined into one tube with 100 uL final volume
2 hrs at 37C

PCR cleanup of AP-treated pSB4K5

Qiagen kit
concentration 45.2 ng/uL in 50 uL

Ligation of pSB4K5 and R0040-anti-tracrRNA

2 experimental plates with 3x and 6x molar insert
pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL
R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL
Backbone-only and insert-only controls
Transformed via heat shock and plated on LB+G418

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

Minipreps

pSB1C3-R0040 (4 copies)

concentrations 47.4, 80.7, 109.7, 193.5

Objective: Switch dCas9-tracrRNA into pSB1C3

Treat pSB1C3 with Antarctic Phosphatase

cleaned copy from 7/28 with 100 uL final volume
2 hrs at 37C

PCR cleanup of AP-treated pSB1C3

Qiagen kit
concentration 104.5 ng/uL in 50 uL

Ligation of pSB1C3 and dCas9-tracrRNA

2 experimental plates

3x molar dCas9-tracrRNA “insert”

pSB1C3 100 ng = 1 uL
dCas9-tracrRNA 600 ng = 7.5 uL

3x molar pSB1C3 “backbone”

pSB1C3 150 ng = 1.5 uL
dCas9-tracrRNA 100 ng = 1.2 uL

Backbone-only and insert-only controls
Transformed via heat shock and plated on LB+Cm

July 31

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Plate results:

Hundreds of colonies on both experimental plates
20-30 colonies on backbone-only control
0 colonies on insert-only control

Cultured colonies

4 copies of pSB4K5-R0040-anti-tracrRNA

Objective: Switch dCas9-tracrRNA and dCas9-tracrRNA-crRNA into pSB1C3

Plate results:

Experimental plate 2 (higher pSB1C3 concentration) had 2 colonies
Experimental plate 1 had no colonies
Backbone-only control had 1 colony, insert-only control had 0

Cultured colonies

2 copies of potential pSB1C3-dCas9-tracrRNA

PCRs of tracrRNA-dCas9 and tracrRNA-dCas9-crRNA

8 tubes of each, with 1 uL 1 ng/uL pdCas9 template per tube
tracrRNA-dCas9 oligos: dCas9tracr-up and dCas9tracr-dn
tracrRNA-dCas9-crRNA oligos: dCas9tracr-up and pdCas9-dn
Q5 polymerase
64C anneal temp, 2:30 extension time

Agarose gel of PCRs

1-8: tracrRNA-dCas9-crRNA
9-16: tracrRNA-dCas9
Results: all 16 lanes look good

PCR cleanup of PCRs

8 tubes into 50 uL for each of two PCRs
Concentrations

tracrRNA-dCas9-crRNA: 350 ng/uL
tracrRNA-dCas9: 335 ng/uL

Prep-scale digest of 2 PCRs

tracrRNA-dCas9-crRNA and tracrRNA-dCas9
Each in 100 uL final volume with 2 uL each of XbaI/PstI/DpnI

PCR cleanup of digests

Into 50 uL final volume

Objective: Put R0040 in front of Csy4 scaffold and Repeat-seq scaffold

Prep-scale digest

pSB1C3-R0040 (2 copies) with 2.5 uL each of SpeI/PstI
100 uL final volume each copy

PCR cleanup of digests

Into 50 uL final volume

Objective: Triple-transformation of R0040-anti-tracrRNA into Z1-reporter-crRNA_GFP cells

Cultured cells in morning

straight from frozen stocks into 2 mL LB+Cm+Amp
DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP1
DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP3

Cultured cells in evening

1 mL from morning culture into 50 mL flask for overnight growth
To make chemically competent cells

Objective: Create pSB1C3-R0011-I13500

Cultured colonies

2 copies of pSB1C3-R0011 from streak plate of original frozen stock

@@ Line 6: / Line 6: @@
 <body>
-<div id="sticky_mj_wrap">
-<div id="cal">
-<table>
+<div class="day">
-<tr>
+<a id="jul1"><h2> July 1 </h2></a>
-<td colspan="7"> May 2014 </td>
+<div class="lab">
-</tr>
+<p class="obj">Objective: make LB+Cm plates </p>
-<tr>
-<td colspan="7" align=center><i> Month 2 of Project </i></td>
+<ul>
-</tr>
+<li>Four sleeves of plates with 4x500mL medium</li>
-<tr>
+<li>34mg/mL chloramphenicol</li>
-<td>sun</td>
+</ul>
-<td>mon</td>
-<td>tue</td>
+<p class="obj">Objective: insert crRNAs into pdCas9 and scaffold</p>
-<td>wed</td>
+<p>Plate results</p>
-<td>thu</td>
-<td>fri</td>
+<ul>
-<td>sat</td>
+<li>Experimental had lots of colonies, but backbone-only control did as well</li>
-</tr>
+</ul>
-<tr>
-<td></td>
+<ul>
-<td></td>
+<li>reasoning: similarity of BsaI overhangs may lead to re-ligation</li>
-<td></td>
+<li>solution: treat with CIP before ligation</li>
-<td></td>
+</ul>
-<td>1</td>
+<p>Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold </p>
-<td>2</td>
-<td>3</td>
+<ul>
-</tr>
+<li>plasmids used in 6/30/14 transformation</li>
-<tr>
+<li>to make sure BsaI is cutting properly</li>
-<td>4</td>
+<li>Results: single band in both appears to be cut DNA</li>
-<td>5</td>
+</ul>
-<td>6</td>
+<!-- https://2014.igem.org/File:GEL_MCF_7-1-14.png or https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png-->
-<td>7</td>
+<a href="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png"></a>
-<td>8</td>
-<td>9</td>
+<p>CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)</p>
-<td>10</td>
-</tr>
+<ul>
-<tr>
+<li>pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP</li>
-<td>11</td>
+<li>Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP</li>
-<td>12</td>
+<li>2 hours at 37C (longer than standard protocol)</li>
-<td>13</td>
+</ul>
-<td>14</td>
-<td>15</td>
+<p>PCR cleanup of pdCas9 and Repeat scaffold CIP treatments</p>
-<td>16</td>
-<td>17</td>
+<ul>
-</tr>
+<li>Final concentrations too low to be of use</li>
-<tr>
+</ul>
-<td>18</td>
-<td>19</td>
+<ul>
-<td>20</td>
+<li>Particularly negligible in pdCas9</li>
-<td>21</td>
+<li>Need to culture and cut more plasmid to try ligation again</li>
-<td>22</td>
+</ul>
-<td>23</td>
+<p>Spread plates from pdCas9 and Repeat scaffold frozen stocks</p>
-<td>24</td>
-</tr>
+<ul>
-<tr>
+<li>Used tube &ldquo;1&rdquo; for repeat scaffold</li>
-<td>25</td>
+<li>Plated on fresh LB+Cm plates</li>
-<td>26</td>
+</ul>
-<td>27</td>
-<td>28</td>
+<ul>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#may29">29</a></td>
+<li>Plates still slightly wet, difficult to spread evenly</li>
-<td>30</td>
+</ul>
-<td>31</td>
-</tr>
+<p class="obj">Objective: make CCEC stock of DH5alpha-ZI strain</p>
-<tr>
+<p>Diluted back 5mL culture in morning</p>
-<td></td>
-<td></td>
+<ul>
-<td></td>
+<li>1mL into 5mL LB+Spec</li>
-<td></td>
+</ul>
-<td></td>
-<td></td>
+<ul>
-<td></td>
+<li>note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable</li>
-</tr>
+</ul>
-</table>
+<p>Inoculated 1 mL culture into each of two 250mL flasks</p>
-<br />
-<table>
+<ul>
-<tr>
+<li>Flasks contain SOC</li>
-<td colspan="7"> June 2014 </td>
+<li>30C shaker for overnight growing</li>
-</tr>
+</ul>
-<tr>
-<td colspan="7" align=center><i> Month 3 of Project </i></td>
-</tr>
-<tr>
-<td>sun</td>
-<td>mon</td>
-<td>tue</td>
-<td>wed</td>
-<td>thu</td>
-<td>fri</td>
-<td>sat</td>
-</tr>
-<tr>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun1">1</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun2">2</a></td>
-<td>3</td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun4">4</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun5">5</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun6">6</a></td>
-<td>7</td>
-</tr>
-<tr>
-<td>8</td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun9">9</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun10">10</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun11">11</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun12">12</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun13">13</a></td>
-<td>14</td>
-</tr>
-<tr>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun15">15</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun16">16</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun17">17</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun18">18</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun19">19</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun20">20</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun21">21</a></td>
-</tr>
-<tr>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun22">22</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun23">23</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun24">24</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun25">25</a></td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun26">26</a></td>
-<td>27</td>
-<td>28</td>
-</tr>
-<tr>
-<td>29</td>
-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun30">30</a></td>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-</tr>
-</table>
 </div>
 </div>
-<div id="rightnb">
 <div class="day">
-<a id="july9"><h2>July 9</h2></a>
+<a id="jul2"><h2> July 2 </h2></a>
+<div class="lab">
+<p class="obj">Objective: Make CCEC stock of DH5alpha-ZI strain</p>
+<p>Made fresh CCMB-80 buffer stock</p>
+<ul>
+<li>precipitate formed during pH adjusting and may have filtered out</li>
+</ul>
+<ul>
+<li>could be detrimental to final product</li>
+</ul>
+<p>Followed iGEM online protocol to make CCEC from Z1 strain</p>
+<ul>
+<li>Overnight culture was much more concentrated than recommended</li>
+</ul>
+<ul>
+<li>OD &gt;1</li>
+<li>Did not have enough SOC to dilute back fully, so diluted back only a bit</li>
+</ul>
+<ul>
+<li>Had to vortex to resuspend cells at two stages of procedure</li>
+<li>Final OD after procedure ~1.6 for 100mL of cells</li>
+<li>Aliquoted 1mL into tubes, froze at -80C</li>
+</ul>
+<p>Next steps: </p>
+<ul>
+<li>Test competence with useful strains containing Lac, Tet promoters</li>
+</ul>
+<p class="obj">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</p>
+<p>Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold</p>
+<ul>
+<li>3 colonies per plate in LB+Cm medium at 37C overnight</li>
+<li>Plates were messy--hard to get individual colonies </li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul3"><h2> July 3 </h2></a>
+<div class="lab">
+<p class="obj">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</p>
+<p>Miniprep cultures from 7/2</p>
+<ul>
+<li>Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL</li>
+<li>Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL</li>
+</ul>
+<p>Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold</p>
+<ul>
+<li>to insert crRNAs</li>
+<li>Digest with BsaI in cutsmart buffer</li>
+<li>50 uL DNA in 100 uL reactions with 7.5 uL BsaI</li>
+<li>3 hours 20 min at 37C</li>
+</ul>
+<p>PCR cleanup of BsaI digest</p>
+<ul>
+<li>final concentrations (in 30 uL each):</li>
+</ul>
+<ul>
+<li>pdCas9: 65.4, 70.5 ng/uL</li>
+<li>Repeat-scaffold: 122.3, 114.8 ng/uL</li>
+</ul>
+<p>CIP treatment of pdCas9 and Repeat-scaffold BsaI digests</p>
+<ul>
+<li>Two digest tubes pooled into one CIP tube for each backbone</li>
+<li>60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer</li>
+<li>1.5 hours at 37C</li>
+</ul>
+<p>PCR cleanup of CIP treatment</p>
+<ul>
+<li>final concentrations (in 30 uL):</li>
+</ul>
+<ul>
+<li>pdCas9: 32.5 ng/uL</li>
+<li>Repeat scaffold 129.9 ng/uL</li>
+</ul>
+<p>Annealed oligos of crRNA inserts for ligation</p>
+<ul>
+<li>3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each</li>
+<li>5uL each oligo heated to ~95C, slowly brought to rt in water bath</li>
+</ul>
+<p>Ligation of crRNA inserts into pdCas9 and Repeat-scaffold</p>
+<ul>
+<li>6 working tubes + 5 controls</li>
+</ul>
+<ul>
+<li>pdCas9 + each crRNA (GFP1, GFP2, or GFP3)</li>
+<li>Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)</li>
+<li>pdCas9 Backbone only control</li>
+<li>Repeat-scaffold Backbone only control</li>
+<li>Insert only controls for each insert</li>
+</ul>
+<ul>
+<li>100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned</li>
+<li>100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned</li>
+<li>1uL annealed oligo insert</li>
+<li>10 uL reactions with 0.5 uL T4 Ligase</li>
+<li>Transformed into DH5alpha and plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</p>
+<p>CCEC Transformation efficiency test</p>
+<ul>
+<li>Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC</li>
+</ul>
+<ul>
+<li>Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng</li>
+</ul>
+<ul>
+<li>Also for use in testing pLac regulation in Z1 strain</li>
+</ul>
+<p>Transformed iGEM kit plate 3-6G into DH5alpha-Z1</p>
+<ul>
+<li>contains pSB1C3-BBa_I13521: pTet-RBS-mRFP</li>
+<li>in order to test regulation of pTet in Z1 strain</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul4"><h2> July 4 </h2></a>
+<div class="lab">
+<p class="obj">Objective: ligate crRNAs into pdCas9 and Repeat-scaffold</p>
+<p>Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:</p>
+<ul>
+<li>~1000 colonies on all experimental plates</li>
+<li>BO control for pdCas9 has ~500 colonies</li>
+<li>BO control for Repeat scaffold has ~1000 colonies</li>
+<li>IO controls all have 0 colonies</li>
+</ul>
+<p>Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone</p>
+<ul>
+<li>12 tubes total in LB+Cm</li>
+<li>None from Repeat-scaffold attempts</li>
+</ul>
+<p class="obj">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</p>
+<p>CCEC Test plate results:</p>
+<ul>
+<li>130 ng: 100s of colonies</li>
+<li>13 ng:~100 colonies</li>
+<li>1.3 ng: ~20 colonies</li>
+<li>0.13 ng: 0 colonies</li>
+<li>0.013 ng: 0 colonies</li>
+<li>Competency ~10 colonies/ng (low but working)</li>
+</ul>
+<p>Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1</p>
+<ul>
+<li>Plate had ~5 colonies</li>
+</ul>
+<p>Cultured colonies for Tet and Lac testing in DH5alpha-Z1</p>
+<ul>
+<li>One colony of pSB1C3-K741002</li>
+<li>One colony of pSB1C3-BBa_I13521</li>
+<li>In LB+Cm</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul5"><h2> July 5 </h2></a>
+<div class="lab">
+<p class="obj">Objective: Insert crRNAs into pdCas9 backbone</p>
+<p>Saved stocks of 12 strains in 15% glycerol at -80C</p>
+<ul>
+<li>4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9</li>
+</ul>
+<p>Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts</p>
+<ul>
+<li>4 tubes each </li>
+<li>Eluted in 50 uL EB, all concentrations 100-200 ng/uL</li>
+</ul>
+<p class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</p>
+<p>Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)</p>
+<ul>
+<li>Culture of I13521 did not grow</li>
+</ul>
+<p>Cultured another colony of pSB1C3-BBa_I13521</p>
+<ul>
+<li>from 7/3 plate</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul6"><h2> July 6 </h2></a>
+<div class="lab">
+<p class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</p>
+<p>Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)</p>
+</div>
+</div>
+<div class="day">
+<a id="jul7"><h2> July 7 </h2></a>
+<div class="lab">
+<p class="obj">Objective: insert crRNAs GFP1,2,3 into pdCas9</p>
+<p>Analytical restriction digest </p>
+<ul>
+<li>12 tubes with potential inserts (4 per crRNA)</li>
+<li>pdCas9 as a control</li>
+<li>BsaI and XbaI in cutsmart for 1.5 hrs</li>
+</ul>
+<p>Agarose gel of restriction digest</p>
+<ul>
+<li>Gel 1:</li>
+</ul>
+<ul>
+<li>1-4: pdCas9-crRNA-GFP1</li>
+<li>5-8: pdCas9-crRNA-GFP2</li>
+<li>9-12: pdCas9-crRNA-GFP3</li>
+<!--https://2014.igem.org/File:GEL_7-6-14_AJC.png or https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png-->
+<li><a href="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png"></a></li>
+</ul>
+<ul>
+<li>Gel 2:</li>
+</ul>
+<ul>
+<li>pdCas9 (control)</li>
+<!--https://2014.igem.org/File:GEL_7-6-14_AJC_2.png or https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png-->
+<li><a href="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png"></a></li>
+</ul>
+<ul>
+<li>Expected single band ~9.3 kb if BsaI cut and ligation worked</li>
+<li>Control and uncut failures expect two bands, 5.0 and 4.2 kb</li>
+<li>Results: all 12 samples have single band</li>
+</ul>
+<ul>
+<li>No uncut plasmids</li>
+<li>Could still be recircularized with no insert</li>
+</ul>
+<p>gel pictures:</p>
+<p>Anneal oligos of 3 crRNA-GFP inserts</p>
+<ul>
+<li>Following Dewran&rsquo;s crRNA protocol</li>
+<li>2 uL each oligo diluted in PBS to 20 uL</li>
+<li>Placed in 98C water bath, cooled gradually to rt</li>
+</ul>
+<p>Phosphorylate oligo inserts of crRNAs</p>
+<ul>
+<li>5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK</li>
+<li>30 mins at 37C, then deactivated for 20 mins at 65C</li>
+<li>Diluted 1/50</li>
+</ul>
+<p>Ligation </p>
+<ul>
+<li>75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9</li>
+</ul>
+<ul>
+<li>Both BsaI cut and CIP treated 7/3/14</li>
+</ul>
+<ul>
+<li>1.5 uL diluted, phosphorylated, annealed oligo insert</li>
+<li>6 experimental tubes + 2 BO controls and 3 IO controls</li>
+<li>Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm</li>
+</ul>
+<p>Cultured 2 colonies from pdCas9 plate</p>
+<ul>
+<li>For Charlie to try his hands at ligation</li>
+</ul>
+<p class="obj">Objective: Prepare various backbones for multiple-plasmid use</p>
+<p>Transformed two distribution kit parts</p>
+<ul>
+<li>Plate 4-6H: pSB4K5-J04450</li>
+</ul>
+<ul>
+<li>backbone contains pSC101 origin with Kan resistance</li>
+</ul>
+<ul>
+<li>Plate 4-18A: pSB3K3-I20260</li>
+</ul>
+<ul>
+<li>backbone contains p15A origin with Kan resistance</li>
+<li>On LB+G418 plates </li>
+</ul>
+<p>Streaked plate from frozen stock</p>
+<ul>
+<li>pSB6A1-J04450</li>
+</ul>
+<ul>
+<li>backbone contains pMB1 origin with Amp resistance</li>
+<li>on LB+Amp</li>
+</ul>
+<p class="obj">Objective: Test pTet and pLac in DH5alphaZ1 strain</p>
+<p>Streaked plates from frozen stock</p>
+<ul>
+<li>pSB1C3-K741002 in DH5alpha-Z1</li>
+</ul>
+<ul>
+<li>contains pLac-GFP</li>
+</ul>
+<ul>
+<li>pSB1C3-I13521 in DH5alpha-ZI</li>
+</ul>
+<ul>
+<li>contains pTet-RFP</li>
+</ul>
+<ul>
+<li>On LB+Cm plates</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul8"><h2> July 8 </h2></a>
+<div class="lab">
+<p class="obj">Objective: insert crRNAs into pdCas9</p>
+<p>Plate results from 7/7 transformation</p>
+<ul>
+<li>pdCas9-crRNAs: ~50 colonies each</li>
+<li>pdCas9 BO: 100s of colonies</li>
+<li>Repeat-crRNAs: 100s of colonies</li>
+<li>Repeat BO: ~1000 colonies</li>
+<li>IO controls: 0 colonies, ~15 for crRNA-GFP3</li>
+<li>Notes: unsure why BO controls have more colonies than experimental</li>
+</ul>
+<p>Restriction digest of 12 samples from 7/5 prep + pdCas9 control</p>
+<ul>
+<li>With SacI/NheI</li>
+</ul>
+<ul>
+<li>Should show difference between insert and recircularized backbone</li>
+</ul>
+<p>Made and ran 1.5% agarose gel in TBE</p>
+<ul>
+<li>for better resolution of small band differences in pdCas9 with/without insert</li>
+</ul>
+<ul>
+<li>1: pdCas9 (control)</li>
+<li>2-5: pdCas9-crRNA-GFP1 (1-4)</li>
+<li>6-9: pdCas9-crRNA-GFP2 (1-4)</li>
+<li>10-13: pdCas9-crRNA-GFP3 (1-4)</li>
+<li>14: pdCas9 (control)</li>
+</ul>
+<ul>
+<li>expected: </li>
+</ul>
+<ul>
+<li>3.1, 2.8, and 2.7 kb bands</li>
+<li>585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)</li>
+</ul>
+<ul>
+<li>Results: One sample, crRNA-GFP3-3, has larger band matching control</li>
+</ul>
+<ul>
+<li>This colony may be correct, should be sequenced</li>
+<li>Others have smaller band, are probably incorrect</li>
+</ul>
+<p>Culture 40 colonies from existing plates to screen for crRNA inserts</p>
+<ul>
+<li>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14</li>
+<li>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14</li>
+<li>in LB+Cm</li>
+</ul>
+<p class="obj">Objective: Prepare various backbones for multiple-plasmid use</p>
+<p>Prepared LB+Kan medium</p>
+<ul>
+<li>50 ug/mL final concentration Kanamycin</li>
+</ul>
+<p>Cultured colonies from transformations and stock streaking</p>
+<ul>
+<li>3 colonies each from 3 plasmids:</li>
+</ul>
+<ul>
+<li>pSB6A1-J04450 in LB+Amp</li>
+<li>pSB3K3-I20260 in LB+Kan</li>
+<li>pSB4K5-J04450 in LB+Kan</li>
+</ul>
+<p class="obj">Objective: Test pLac and pTet in DH5alpha-Z1</p>
+<p>Notes:</p>
+<ul>
+<li>Need to obtain aTc for pTet testing</li>
+</ul>
+<ul>
+<li>Tried to use Doxycycline, but had trouble with solubility in ethanol (?)</li>
+</ul>
+<ul>
+<li>pLac is not the final promoter to be used, a different variant will be used</li>
+</ul>
+<ul>
+<li>with no glucose dependency</li>
+</ul>
+<ul>
+<li>Flow testing of pLac and pTet was suspended until these changes are made</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul9"><h2>July 9</h2></a>
 <div class="obj">Objective: Insert crRNAs into pdCas9</div>
 <div class="lab">
@@ Line 275: / Line 677: @@
 <div class="day">
-<a id="july10"><h2>July 10</h2></a>
+<a id="jul10"><h2>July 10</h2></a>
 <div class="obj">Objective: Obtain backbones and switch parts into new backbones</div>
 <div class="lab">
@@ Line 344: / Line 746: @@
 <div class="day">
-<a id="july11"><h2>July 11</h2></a>
+<a id="jul11"><h2>July 11</h2></a>
 <div class="obj">Objective: Switch K608012 into pSB6A1</div>
 <div class="lab">
@@ Line 475: / Line 877: @@
 <div class="day">
-<a id="july14"><h2>July 14</h2></a>
+<a id="jul14"><h2>July 14</h2></a>
 <div class="obj">Objective: insert crRNAs into pdCas9</div>
 <div class="lab">
@@ Line 621: / Line 1,023: @@
 <div class="day">
-<a id="july15"><h2>July 15</h2></a>
+<a id="jul15"><h2>July 15</h2></a>
 <div class="obj">Objective: Create pTet-anti-tracrRNA</div>
 <div class="lab">
@@ Line 735: / Line 1,137: @@
 <div class="day">
-<a id="july16"><h2>July 16</h2></a>
+<a id="jul16"><h2>July 16</h2></a>
 <div class="obj">Objective: Create pTet-anti-tracrRNA</div>
 <div class="lab">
@@ Line 833: / Line 1,235: @@
-<span>7/17/14</span></h2><p class="c6"><span class="c0 c3">Objective: Create pTet-anti-tracrRNA and pSB1C3-R0011-I13500</span></p><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Minipreps </span></p><ul class="c7 lst-kix_jwintdkqr7vg-0 start"><li class="c1"><span class="c0">4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation</span></li></ul><ul class="c7 lst-kix_8jaag76tmmre-0 start"><li class="c1"><span class="c0">4 copies of pSB1C3-R0011-I13500 from 7/15 ligation</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Analytical digest of minipreps</span></p><ul class="c7 lst-kix_lxs7wstahkp3-0 start"><li class="c1"><span class="c0">pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI</span></li><li class="c1"><span class="c0">pSB1C3-R0011-I13500 with MfeI/NdeI/SacI</span></li></ul><p class="c6 c23"><span class="c0 c4">Agarose gel of digest results:</span></p><ul class="c7 lst-kix_vio5o0mh9rst-0 start"><li class="c1"><span class="c0">Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, &lt;0.1 kb bands)</span></li><li class="c1"><span class="c0">Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)</span></li><li class="c1"><span class="c0 c4">Results: </span></li></ul><ul class="c7 lst-kix_vio5o0mh9rst-1 start"><li class="c2"><span class="c0 c4">R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly</span></li><li class="c2"><span class="c0 c4">R0011-I13500 does not look correct (only one band at 2.0 kb)</span></li></ul><p class="c6"><span class="c0 c3">Objective: Switch dCas9-tracrRNA into pSB1C3</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Colony PCR on pSB1C3-dCas9-tracrRNA plate</span></p><ul class="c7 lst-kix_3muxqzmhmgsp-0 start"><li class="c1"><span class="c0">Ligation plate from 7/15/14</span></li><li class="c1"><span class="c0">15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC</span></li><li class="c1"><span class="c0">oligos SB1C3-up1 and SB1C3-dn1</span></li><li class="c1"><span class="c0">annealed at 65C, extension 2 mins</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of colony PCR</span></p><ul class="c7 lst-kix_nxjmmijl9oy9-0 start"><li class="c1"><span class="c0 c4">results: primer-bands only (no successes)</span></li></ul><p class="c6"><span class="c0 c3">Objective: Switch K608012 into pSB6A1</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Culture colonies</span></p><ul class="c7 lst-kix_6jc4x55ftesi-0 start"><li class="c1"><span class="c0">2 copies of pSB6A1-J04450 (from 6-9-14 plate)</span></li><li class="c1"><span class="c0">2 copies of pSB1C3-K608012</span>
 <div class="day">
-<a id=""><h2></h2></a>
+<a id="jul17"><h2>July 17</h2></a>
-<div class="obj"></div>
+<div class="obj">Objective: Create pTet-anti-tracrRNA and pSB1C3-R0011-I13500</div>
 <div class="ppl"></div>
 <div class="lab">
-<p></p>
+<p>Minipreps</p>
 <ul>
-<li></li>
+<li>4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation</li>
 </ul>
+<ul>
+<li>4 copies of pSB1C3-R0011-I13500 from 7/15 ligation</li>
+</ul>
+<p>Analytical digest of minipreps</p>
+<ul>
+<li>pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI</li>
+<li>pSB1C3-R0011-I13500 with MfeI/NdeI/SacI</li>
+</ul>
+<p>Agarose gel of digest results:</p>
+<ul>
+<li>Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, &lt;0.1 kb bands)</li>
+<li>Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)</li>
+<li>Results: </li>
+</ul>
+<ul>
+<li>R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly</li>
+<li>R0011-I13500 does not look correct (only one band at 2.0 kb)</li>
+</ul>
+<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
+<p>Colony PCR on pSB1C3-dCas9-tracrRNA plate</p>
+<ul>
+<li>Ligation plate from 7/15/14</li>
+<li>15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC</li>
+<li>oligos SB1C3-up1 and SB1C3-dn1</li>
+<li>annealed at 65C, extension 2 mins</li>
+</ul>
+<p>Agarose gel of colony PCR</p>
+<ul>
+<li>results: primer-bands only (no successes)</li>
+</ul>
+<div class="obj">Objective: Switch K608012 into pSB6A1</div>
+<p>Culture colonies</p>
+<ul>
+<li>2 copies of pSB6A1-J04450 (from 6-9-14 plate)</li>
+<li>2 copies of pSB1C3-K608012
+</p>
 </div>
 </div>
-<!-- Comment here -->
-<!-- To insert a picture, the command is <img src=""> -->
+<div class="day">
+<a id="jul18"><h2>July 18</h2></a>
+<div class="lab">
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>PCR of dCas9-tracrRNA from pdCas9</p>
+<ul>
+<li>8 tubes, each with 1 uL pdCas9 template at 1 ng/uL</li>
+<li>oligos dCas9tracr-up and dCas9-tracr-dn</li>
+<li>2 min extension, 64C anneal</li>
+</ul>
+<p>Agarose gel of PCR product</p>
+<ul>
+<li>All 8 tubes look successful (band ~4 kb)</li>
+</ul>
+<p>PCR cleanup of dCas9-tracrRNA</p>
+<ul>
+<li>Qiagen kit</li>
+<li>Into 2 tubes, 30 uL each</li>
+</ul>
+<p>Prep-scale digest of PCR product</p>
+<ul>
+<li>with PstI/XbaI/DpnI</li>
+<li>in 100 uL each for 3 hrs at 37C</li>
+</ul>
+<p>Treat pSB1C3 digest with CIP</p>
+<ul>
+<li>1 hr at 37C</li>
+</ul>
+<p class="obj">Objective: Switch K608012 into pSB6A1</p>
+<p>Miniprep pSB1C3-K608012 and pSB6A1-J04450</p>
+<ul>
+<li>1 copy each (the second copy of each did not grow in culture)</li>
+<li>concentrations:</li>
+</ul>
+<ul>
+<li>pSB1C3-K608012: 344 ng/uL</li>
+<li>pSB6A1-J04450: 421.5 ng/uL</li>
+</ul>
+<p>Prep-scale digest of K608012 and pSB6A1</p>
+<ul>
+<li>50 uL DNA in 100 uL reaction</li>
+<li>Both with EcoRI and PstI</li>
+<li>3 hrs at 37C</li>
+</ul>
+<p>CIP treatment of pSB6A1 and heat-inactivation</p>
+<ul>
+<li>2.5 uL CIP added to digest tube</li>
+<li>Both digests returned to 37C for 1 hr</li>
+<li>Then 30 mins at 80C to heat-inactivate</li>
+</ul>
+<p>Gel extraction of pSB6A1 and K608012</p>
+<ul>
+<li>Gel had no EtBr added, and was discarded (product was lost)</li>
+</ul>
 </div>
+</div>
+<div class="day">
+<a id="jul20"><h2>July 20</h2></a>
+<div class="lab">
+<p class="obj">Objective: culture colonies for various purposes</p>
+<p>Cultured colonies</p>
+<ul>
+<li>4 copies pSB6A1-J04450</li>
+</ul>
+<ul>
+<li>2 from older plate, 2 from newer</li>
+</ul>
+<ul>
+<li>4 copies pSB1C3-K608012</li>
+</ul>
+<ul>
+<li>2 from older plate, 2 from newer</li>
+</ul>
+<ul>
+<li>4 copies pSB1C3-B0030</li>
+</ul>
+<ul>
+<li>To obtain pSB1C3 without gel extraction</li>
+</ul>
+<ul>
+<li>4 copies pSB1C3-R0011</li>
+<li>4 copies pSB1C3-I13500</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul21"><h2>July 21</h2></a>
+<div class="lab">
+<p class="obj">Objective: Sequence to confirm constructs</p>
+<p>Sequencing reaction and Order #1634</p>
+<ul>
+<li>1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1</li>
+<li>5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1</li>
+<li>9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1</li>
+<li>11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1</li>
+<li>Big Dye, buffer version 1.1</li>
+</ul>
+<p>Order 1634 Results:</p>
+<ul>
+<li>pdCas9-GFP3 copies 3, 6, and 8 are correct</li>
+</ul>
+<ul>
+<li>copy 5 has a single mutation</li>
+</ul>
+<ul>
+<li>anti-tracrRNA: both copies are correct</li>
+</ul>
+<ul>
+<li>copy 1 was a cleaner, more reliable </li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Minipreps</p>
+<ul>
+<li>Qiagen kit</li>
+<li>4 tubes of pSB1C3-R0011</li>
+</ul>
+<ul>
+<li>concentrations 255.7, 169.2, 204.8, 151.2 ng/uL</li>
+</ul>
+<ul>
+<li>4 tubes of pSB1C3-I13500</li>
+</ul>
+<ul>
+<li>concentrations 148.9, 214.2, 260.7, 432.5</li>
+</ul>
+<p>Prep scale digests of pSB1C3-R0011 and I13500</p>
+<ul>
+<li>pSB1C3-R0011 with SpeI/PstI, samples 1,3</li>
+<li>pSB1C3-I13500 with XbaI/PstI, samples 1,3</li>
+<li>50 uL DNA in 100 uL each reaction, 2 tubes per construct</li>
+<li>3 hrs at 37C</li>
+<li>Added CIP (2.5 uL) to pSB1C3-R0011 reaction</li>
+<li>1 more hr for both at 37C</li>
+<li>Heat inactivation of both reactions: 30 mins at 80C</li>
+</ul>
+<p>Gel extraction of I13500</p>
+<ul>
+<li>extraction gel 1, lanes 1 and 2</li>
+<li>Zymoclean prep kit</li>
+<li>sample 1 (lane 1): 300 mg gel yields</li>
+<li>sample 3 (lane 2): 550 mg gel yields</li>
+</ul>
+<p>PCR cleanup of pSB1C3-R0011 </p>
+<ul>
+<li>Qiagen kit</li>
+<li>Two tubes of digest combined into one tube product</li>
+<li>Concentration </li>
+</ul>
+<p>Ligation of pSB1C3-R0011 and I13500</p>
+<ul>
+<li>Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL</li>
+<li>New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL</li>
+<li>Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL</li>
+<li>New insert (I13500 extracted 7/21): 300 ng = 3 uL</li>
+<li>Ligations numbered 1-8:</li>
+</ul>
+<ul>
+<li>1. OB+OI</li>
+<li>2. OB+NI</li>
+<li>3. NB+OI</li>
+<li>4. NB+NI</li>
+<li>5. OB control</li>
+<li>6. OI control</li>
+<li>7. NB control</li>
+<li>8. NI control</li>
+</ul>
+<ul>
+<li>1 hr at rt</li>
+<li>Transformed via heat shock into DH5alphas, plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>Minipreps</p>
+<ul>
+<li>Qiagen kit</li>
+<li>4 tubes of pSB1C3-B0030</li>
+</ul>
+<ul>
+<li>to obtain pSB1C3 without gel extraction</li>
+<li>concentrations 116.8, 99.9, 115.3, 145.1 ng/uL</li>
+</ul>
+<p>PCR cleanup of dCas9-tracrRNA PCR</p>
+<ul>
+<li>Product digested 7/18 with XbaI/PstI/DpnI</li>
+<li>Concentration 80.5 ng/uL in 30 uL</li>
+<li>Heat inactivation of cleaned product, 30 mins at 80C</li>
+</ul>
+<p>Prep scale digest of pSB1C3-B0030</p>
+<ul>
+<li>With XbaI/PstI, samples 1,3</li>
+<li>50 uL DNA in 100 uL each reaction, 2 tubes </li>
+<li>3 hrs at 37C</li>
+<li>Added CIP (2.5 uL) </li>
+<li>1 more hr for both at 37C</li>
+<li>Heat inactivation: 30 mins at 80C</li>
+</ul>
+<p>PCR cleanup of pSB1C3 </p>
+<ul>
+<li>B0030 should not be retained</li>
+<li>Qiagen kit</li>
+<li>Two tubes of digest combined into one tube product</li>
+<li>Concentration </li>
+</ul>
+<p>Ligation of pSB1C3 and dCas9-tracrRNA</p>
+<ul>
+<li>Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL</li>
+<li>Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL</li>
+<li>New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL</li>
+<li>Ligations numbered 9-13:</li>
+</ul>
+<ul>
+<li>9. B+OI</li>
+<li>10. B+NI</li>
+<li>11. B control</li>
+<li>12. OI control</li>
+<li>13. NI control</li>
+</ul>
+<ul>
+<li>1 hr at rt</li>
+<li>Transformed via heat shock into DH5alphas, plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Switch K608012 into pSB6A1 </p>
+<p>Minipreps</p>
+<ul>
+<li>Qiagen kit</li>
+<li>4 tubes of pSB6A1-J04450</li>
+</ul>
+<ul>
+<li>concentrations 189.3, 432.5, 688.7, 487.3 ng/uL</li>
+</ul>
+<ul>
+<li>4 tubes of pSB1C3-K608012</li>
+</ul>
+<ul>
+<li>concentrations 68.8, 91.6, 346.1, ng/uL</li>
+</ul>
+<p>Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012</p>
+<ul>
+<li>pSB6A1-J04450 with XbaI/SpeI, samples 1,3</li>
+<li>pSB1C3-K608012 with EcoRI/PstI, samples 1,3</li>
+<li>50 uL DNA in 100 uL each reaction, 2 tubes per construct</li>
+<li>3 hrs at 37C</li>
+<li>Added CIP (2.5 uL) to pSB6A1-J04450 reaction</li>
+<li>1 more hr for both at 37C</li>
+<li>Heat inactivation of both reactions: 30 mins at 80C</li>
+</ul>
+<p>Gel extraction of pSB6A1 and K608012</p>
+<ul>
+<li>K608012: gel 1, lane 3 and gel 2, lane 1</li>
+<li>pSB6A1: gel 2, lanes 2 and 3</li>
+<li>Zymoclean prep kit</li>
+<li>K608012 sample 1 (lane 1-3): 300 mg gel yields</li>
+<li>K608012 sample 3 (lane 2-1): 550 mg gel yields</li>
+<li>pSB6A1 sample 1 (lane 2-2):</li>
+<li>pSB6A1 sample 3 (lane 2-3):</li>
+</ul>
+<p>Ligation of pSB6A1 and K608012</p>
+<ul>
+<li>Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL</li>
+<li>Old insert (K608012 extracted 7/15): 300 ng = 4 uL</li>
+<li>Ligations numbered 14-16:</li>
+</ul>
+<ul>
+<li>14. B+I</li>
+<li>15. B control</li>
+<li>16. I control</li>
+</ul>
+<ul>
+<li>1 hr at rt</li>
+<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
+</ul>
+<p>Gibson of pSB6A1 and K608012</p>
+<ul>
+<li>New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</li>
+<li>New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</li>
+<li>Numbered 17-19:</li>
+</ul>
+<ul>
+<li>17. B+I</li>
+<li>18. B control</li>
+<li>19. I control</li>
+</ul>
+<ul>
+<li>15 uL Mert&rsquo;s DIY Gibson mix</li>
+<li>1 hr at 60C</li>
+<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
+</ul>
+<p>SLIC of pSB6A1 and K608012</p>
+<ul>
+<li>New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</li>
+<li>New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</li>
+<li>Numbered 20-22:</li>
+</ul>
+<ul>
+<li>20. B+I</li>
+<li>21. B control</li>
+<li>22. I control</li>
+</ul>
+<ul>
+<li>2.5 mins at rt, then 10 mins at 4C</li>
+<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul22"><h2>July 22</h2></a>
+<div class="lab">
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Plate results:</p>
+<ul>
+<li>All four experimental plates had lots of colonies</li>
+<li>both Backbone-only controls had similar colony numbers to experimentals</li>
+<li>Insert-only controls had no colonies</li>
+</ul>
+<p>Colony PCR</p>
+<ul>
+<li>4 copies each of 4 experimental plates = 16 colonies</li>
+<li>Oligos SB1C3-up and SB1C3-dn with Taq polymerase</li>
+<li>66C anneal temp, 2 min extension time</li>
+</ul>
+<p>Agarose gel of colony PCR results</p>
+<ul>
+<li>1-4: OB+OI pSB1C3-R0011-I13500</li>
+<li>5-8: OB+NI pSB1C3-R0011-I13500</li>
+<li>9-12: NB+OI pSB1C3-R0011-I13500</li>
+<li>13-16: NB+NI pSB1C3-R0011-I13500</li>
+<li>17-20: B+OI pSB1C3-dCas9-tracrRNA</li>
+<li>21-24: B+NI pSB1C3-dCas9-tracrRNA</li>
+</ul>
+<ul>
+<li>Results: no successful bands on any colonies</li>
+</ul>
+<ul>
+<li>Just primer-dimers or small amplified bands?</li>
+</ul>
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>Plate results:</p>
+<ul>
+<li>Both experimental plates had lots of colonies</li>
+<li>Backbone-only controls had similar colony numbers to experimentals</li>
+<li>Insert-only controls had no colonies</li>
+</ul>
+<p>Colony PCR</p>
+<ul>
+<li>4 copies each of 2 experimental plates = 8 colonies</li>
+<li>Oligos SB1C3-up and SB1C3-dn with Taq polymerase</li>
+<li>66C anneal temp, 2 min extension time</li>
+</ul>
+<p>Agarose gel of colony PCR results</p>
+<ul>
+<li>see above for gel order</li>
+</ul>
+<ul>
+<li>Results: no successful bands on any colonies</li>
+</ul>
+<ul>
+<li>Just primer-dimers or small amplified bands?</li>
+</ul>
+<p class="obj">Objective: Switch K608012 into pSB6A1</p>
+<p>Plate results:</p>
+<ul>
+<li>No colonies on Gibson or SLIC plates</li>
+<li>3 colonies on Ligation plate</li>
+<li>A few colonies on controls, but not many</li>
+</ul>
+<p>Cultured colonies</p>
+<ul>
+<li>3 colonies of potential pSB6A1-K608012 from Ligation plate</li>
+<li>in LB+Amp</li>
+</ul>
+<p>Ligation of K608012 and pSB6A1</p>
+<ul>
+<li>Same constructs as 7/21 ligation</li>
+<li>This time let recover in SOC for 2 hrs instead of 1 hr</li>
+<li>Plated on LB+Amp</li>
+</ul>
+<p class="obj">Objective: Try putting K608012 into pSB1A2 instead</p>
+<p>Transformation from kit plate</p>
+<ul>
+<li>Plate 4-1N</li>
+</ul>
+<ul>
+<li>pSB1A2-B0034</li>
+</ul>
+<ul>
+<li>Plated on LB+Amp</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul23"><h2>July 23</h2></a>
+<div class="lab">
+<p class="obj">Objective: Switch K608012 into pSB6A1</p>
+<p>Miniprep 3 copies of potential pSB6A1-K608012</p>
+<ul>
+<li>Qiagen kit</li>
+<li>Saved stocks in 15% glycerol</li>
+</ul>
+<p>Analytical digest of minipreps</p>
+<ul>
+<li>With NdeI in Cutsmart, 10 uL final volume</li>
+</ul>
+<p>Agarose gel of analytical digest</p>
+<ul>
+<li>Results:</li>
+</ul>
+<ul>
+<li>Copies 1 and 2 are strange and incorrect</li>
+<li>Copy 3 looks correct</li>
+<li>Need to check with more digests</li>
+</ul>
+<p>Analytical digest to check promising sample</p>
+<ul>
+<li>Copy 3 in 3 different reactions</li>
+</ul>
+<ul>
+<li>EcoRI/SpeI</li>
+<li>EcoRI/MfeI</li>
+<li>NcoI/PvuI</li>
+</ul>
+<ul>
+<li>Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI</li>
+<li>Control: pSB1C3-K608012 with MfeI/NcoI</li>
+</ul>
+<p>Agarose gel of analytical digest</p>
+<ul>
+<li>1. Ligation 3 with EcoRI/SpeI</li>
+<li>2. Ligation 3 with EcoRI/MfeI</li>
+<li>3. Ligation 3 with NcoI/PvuI</li>
+<li>4. pSB6A1-J04450 with EcoRI/SpeI/PvuI</li>
+<li>5. pSB1C3-K608012 with MfeI/NcoI</li>
+</ul>
+<ul>
+<li>Results:</li>
+</ul>
+<ul>
+<li>Sample looks good</li>
+<li>Incorrect samples 1 and 2 glycerol stocks thrown out</li>
+</ul>
+<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p>
+<p>Treat pSB4K5 with CIP</p>
+<ul>
+<li>1.25 uL CIP in 25 uL reaction</li>
+<li>pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9</li>
+</ul>
+<p>Prep-scale digest to obtain R0040-anti-tracrRNA</p>
+<ul>
+<li>pSB1C3-R0040-anti-tracrRNA </li>
+<li>with EcoRI/SpeI</li>
+</ul>
+<p>Gel extraction and cleanup of R0040-anti-tracrRNA</p>
+<ul>
+<li>Extracted smaller band</li>
+<li>Attempted to clean up using Qiagen prep kit</li>
+</ul>
+<ul>
+<li>dissolved with Zymoclean ADB</li>
+<li>Then followed Qiagen PCR cleanup protocol</li>
+<li>510 mg gel yielded little or no DNA </li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Colony PCR</p>
+<ul>
+<li>8 colonies from 7/21 ligation plates</li>
+<li>Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011</li>
+</ul>
+<p>Agarose gel of colony PCR results</p>
+<ul>
+<li>No successes</li>
+<li>Controls did not work properly either</li>
+<li>colony PCR does not seem to be effective--need to just pick colonies</li>
+</ul>
+<p>Culture colonies</p>
+<ul>
+<li>8 colonies of potential pSB1C3-R0011-I13500</li>
+<li>From 7/21 ligation plates (colonies from today&rsquo;s colony PCR)</li>
+</ul>
+<p class="obj">Objective: Measurement interlab study</p>
+<p>Transformations from kit plates</p>
+<ul>
+<li>Plate 1-20K</li>
+</ul>
+<ul>
+<li>pSB1C3-K823005</li>
+</ul>
+<ul>
+<li>Plate 1-22I</li>
+</ul>
+<ul>
+<li>pSB1C3-K823012</li>
+</ul>
+<ul>
+<li>Plate 2-24B</li>
+</ul>
+<ul>
+<li>pSB1C3-E0240</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul24"><h2>July 24</h2></a>
+<div class="lab">
+<!--
+GEL 7-24-14_AJC.png
+-->
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Miniprep 8 colonies of potential pSB1C3-R0011-I13500</p>
+<ul>
+<li>Qiagen kit</li>
+</ul>
+<p>Analytical digest of minipreps</p>
+<ul>
+<li>8 copies with NcoI</li>
+<li>8 copies with EcoRI/SpeI</li>
+</ul>
+<p>Agarose gel of digest results</p>
+<ul>
+<li>1-8: copies 1-8 with NcoI</li>
+<li>9-16: copies 1-8 with EcoRI/SpeI</li>
+<li>Results: no successes, insert appears to be missing</li>
+</ul>
+<ul>
+<li>Unsure whether incorrect colonies are pSB1C3 or pSB1C3-R0011</li>
+</ul>
+<p>Agarose gel of digest results</p>
+<ul>
+<li>Thicker gel, longer run time, more DNA</li>
+<li>from same digest tubes</li>
+<li>Results: </li>
+</ul>
+<ul>
+<li>no small band on EcoRI/SpeI cut and no difference in larger band for the two cuts, indicating that R0011 is not present</li>
+</ul>
+<p>Ligation of pSB1C3-R0011 and I13500</p>
+<ul>
+<li>Used lab stock of T4 Ligase and buffer rather than iGEM stock</li>
+<li>100 ng pSB1C3-R0011 = 2.5 uL</li>
+<li>300 ng I13500 = 3 uL</li>
+<li>4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins</li>
+<li>BO and IO controls for each time</li>
+<li>Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)</li>
+</ul>
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>Ligation of pSB1C3 and dCas9-tracrRNA </p>
+<ul>
+<li>Used lab stock of T4 Ligase and buffer rather than iGEM stock</li>
+<li>300 ng pSB1C3 = 3 uL</li>
+<li>100 ng dCas9-tracrRNA PCR = 1 uL</li>
+<li>4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins</li>
+<li>BO and IO controls for each time</li>
+<li>Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)</li>
+</ul>
+<p class="obj">Objective: Test GFP repression by crRNAs </p><p>Transform pSB6A1-K608012 into DH5alpha-ZI</p>
+<ul>
+<li>Plated on LB+Amp</li>
+</ul>
+<p class="obj">Objective: Obtain constructs for various uses</p><p>Streaked plate from frozen stock</p>
+<ul>
+<li>One LB+Cm plate in thirds:</li>
+</ul>
+<ul>
+<li>pSB1C3-R0040-antitracrRNA</li>
+<li>pSB1C3-Repeat-seq scaffold</li>
+<li>pSB1C3-Csy4-gRNA scaffold</li>
+</ul>
+<p class="obj">Objective: Measurement interlab study</p>
+<p>Cultured colonies (2 each)</p>
+<ul>
+<li>pSB1C3-E0240</li>
+<li>pSB1C3-K823005</li>
+<li>pSB1C3-K823012</li>
+<li>pSB3K3-I20260</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul25"><h2>July 25</h2></a>
+<div class="lab">
+<!--
+GEL 7-25-14_1_MCF.png
+GEL 7-25-14_2_MCF.png
+-->
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Plate results:</p>
+<ul>
+<li>no apparent difference between 15, 30, 45, or 60 minute ligations</li>
+<li>many colonies on all Experimental and BO control plates</li>
+<li>no colonies on IO control</li>
+</ul>
+<p>Colony PCR:</p>
+<ul>
+<li>16 colonies from pSB1C3-R0011-I13500 plates</li>
+</ul>
+<ul>
+ <li>4 from each ligation time</li>
+</ul>
+<ul>
+<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
+<li>66C anneal temp, 45 sec extension</li>
+</ul>
+<p>Agarose gel of colony PCR</p>
+<ul>
+<li>No successes</li>
+<li>It seems as though colony PCRs are not working--try control with a gradient</li>
+</ul>
+<p>Gradient Colony PCR</p>
+<ul>
+<li>to optimize with SB1C3-up and SB1C3-dn primers</li>
+<li>template colonies of pSB1C3-K608011 from plate</li>
+<li>anneal temps 62-72 C, extension time 45 sec</li>
+</ul>
+<p>Agarose gel of gradient colony PCR</p>
+<ul>
+<li>left to right: 62C to 72C anneal temps</li>
+<li>Results: 62C was the best anneal temp--worked well</li>
+</ul>
+<ul>
+<li>gradual decrease as temp increased, no amplification above 65C</li>
+</ul>
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>Plate results:</p>
+<ul>
+<li>no apparent difference between 15, 30, 45, or 60 minute ligations</li>
+<li>many colonies on all Experimental and BO control plates</li>
+<li>no colonies on IO control</li>
+</ul>
+<p>Colony PCR:</p>
+<ul>
+<li>16 colonies from pSB1C3-dCas9-tracrRNA plates</li>
+</ul>
+<ul>
+<li>4 from each ligation time</li>
+</ul>
+<ul>
+<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
+<li>66C anneal temp, 45 sec extension</li>
+</ul>
+<p>Agarose gel of colony PCR</p>
+<ul>
+<li>No successes</li>
+<li>It seems as though colony PCRs are not working--try control with a gradient</li>
+</ul>
+<ul>
+<li>see gradient results above</li>
+</ul>
+<p class="obj">Objective: Various ligations</p>
+<p>Prep-scale digests</p>
+<ul>
+<li>pSB1C3-Csy4-scaffold (2 copies) with XbaI/PstI</li>
+</ul>
+<ul>
+<li>to ligate into R0040</li>
+</ul>
+<ul>
+<li>pSB1C3-Repeat-seq scaffold (2 copies) with XbaI/PstI</li>
+</ul>
+<ul>
+<li>to ligate into R0040</li>
+</ul>
+<ul>
+<li>pSB1C3-R0040-anti-tracrRNA (2 copies) with XbaI/PstI</li>
+<li>100 uL reactions each tube</li>
+</ul>
+<p>PCR cleanups on all digests</p>
+<ul>
+<li>Qiagen kit</li>
+<li>Concentrations (in 50 uL each):</li>
+</ul>
+<ul>
+<li>pSB1C3-Csy4-scaffold: 55.9, 105.5 ng/uL</li>
+<li>pSB1C3-Repeat-seq scaffold: 15.2, 55.2 ng/uL</li>
+<li>pSB1C3-R0040-anti-tracrRNA: 90.6 ng/uL</li>
+</ul>
+<ul>
+<li>Need to extract from gels before using, only cleaned up for safer storage </li>
+</ul>
+<p class="obj">Objective: Measurement interlab study</p>
+<p>Minipreps</p>
+<ul>
+<li>Qiagen kit</li>
+</ul>
+<ul>
+<li>pSB1C3-K823005 (2 copies) </li>
+</ul>
+<ul>
+<li>concentrations 256.3, 314.8 ng/uL</li>
+</ul>
+<ul>
+<li>pSB1C3-K823012 (2 copies)</li>
+</ul>
+<ul>
+<li>concentrations 204.4, 192.5 ng/uL</li>
+</ul>
+<ul>
+<li>pSB1C3-E0240 (2 copies)</li>
+</ul>
+<ul>
+<li>concentrations 266.3, 241.5 ng/uL</li>
+</ul>
+<p>Prep-scale digests</p>
+<ul>
+<li>pSB1C3-K823005 (2 copies) with SpeI/PstI</li>
+<li>pSB1C3-K823012 (2 copies) with SpeI/PstI</li>
+<li>pSB1C3-E0240 (2 copies) with XbaI/PstI</li>
+<li>100 uL each tube</li>
+</ul>
+<p>PCR cleanups on all digests</p>
+<ul>
+<li>Qiagen kit</li>
+<li>Concentrations (in 50 uL each):</li>
+</ul>
+<ul>
+<li>pSB1C3-K823005: 156.3, 181.7 ng/uL</li>
+<li>pSB1C3-K823012: 110.0, 98.0 ng/uL</li>
+<li>pSB1C3-E0240: 110.0, 114.6 ng/uL</li>
+</ul>
+<p class="obj">Objective: Test crRNA repression of GFP reporter</p>
+<p>Cultured colony</p>
+<ul>
+<li>To make CCEC of reporter strain</li>
+<li>One colony of pSB6A1-K608012 in DH5alpha-ZI</li>
+<li>Grew for 8 hrs during the day in 2 mL LB+Amp at 37C</li>
+<li>Inoculated into 100 mL flask of LB+Amp for overnight shaker at rt</li>
+</ul>
+<p>Flow cytometry of reporter strain</p>
+<ul>
+<li>quick check to make sure reporter still works with new backbone</li>
+<li>Used parameters from previous GFP flow data</li>
+<li>Levels slightly under what was seen before, but still strong</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul26"><h2>July 26</h2></a>
+<div class="lab">
+<p class="obj">Objective: Test crRNA repression of GFP reporter</p>
+<p>Prepared Chemically competent E. coli </p>
+<ul>
+<li>Using DH5alpha-ZI + pSB6A1-K608012</li>
+<li>iGEM CCEC protocol with smaller volumes (made ~20 1-mL aliquots)</li>
+</ul>
+<p>Double-Transformation of pdCas9 variants into reporter cells</p>
+<p class=”ppl”>Charlie Cooper</p>
+<ul>
+<li>pdCas9 (with BsaI insert as crRNA sequence)</li>
+<li>pdCas9-crRNA_GFP1</li>
+<li>pdCas9-crRNA_GFP2</li>
+<li>pdCas9-crRNA_GFP3</li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Colony PCRs</p>
+<ul>
+<li>16 colonies from 7/24 ligation plates</li>
+<li>oligos SB1C3-up and SB1C3-dn</li>
+<li>62C anneal (as determined by gradient), 45 sec extension</li>
+</ul>
+<p>Agarose gel of Colony PCRs</p>
+<ul>
+<li>Results: colonies 5 and 14 amplified at the correct size</li>
+</ul>
+<p>Culture colonies</p>
+<ul>
+<li>Colonies 5 and 14 from colony PCR of potential pSB1C3-R0011-I13500</li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-dCas9-tracrRNA</p>
+<p>Colony PCRs</p>
+<ul>
+<li>16 colonies from 7/24 ligation plates</li>
+<li>oligos SB1C3-up and SB1C3-dn</li>
+<li>62C anneal (as determined by gradient), 45 sec extension</li>
+<li>PCRs left in machine without starting reaction for ~1 hr, could have kill reaction</li>
+</ul>
+<p>Agarose gel of Colony PCRs</p>
+<ul>
+<li>No successes</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul27"><h2>July 27</h2></a>
+<div class="lab">
+<p class="obj">Objective: Test crRNA repression of GFP reporter</p>
+<p>Cultured colonies for flow</p>
+<ul>
+<li>3 copies of each</li>
+<li>DH5alpha-Z1 (background)</li>
+<li>DH5-alpha-Z1 + pSB6A1-K608012 (reporter positive control)</li>
+<li>DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9 (non-target control)</li>
+<li>DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP1</li>
+<li>DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP2</li>
+<li>DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP3</li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Minipreps</p>
+<ul>
+<li>Colonies 5 and 14 from 7/26 colony PCRs</li>
+<li>Qiagen kit</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul28"><h2>July 28</h2></a>
+<div class="lab">
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Analytical digest of minipreps</p>
+<ul>
+<li>Colonies 5 and 14 of potential pSB1C3-R0011-I13500</li>
+<li>with MfeI</li>
+<li>with NcoI</li>
+<li>with EcoRI/PstI</li>
+</ul>
+<p>Agarose gel of analytical digest results</p>
+<ul>
+<li>Gel Order:</li>
+</ul>
+<ul>
+<li>#14 w/ MfeI</li>
+<li>#14 w/ EcoRI/PstI</li>
+<li>#14 w/ NcoI</li>
+<li>#5 w/ MfeI</li>
+<li>#5 w/ EcoRI/PstI</li>
+<li>#5 w/ NcoI</li>
+</ul>
+<ul>
+<li>Results: All looks correct, except that MfeI cut once instead of twice</li>
+</ul>
+<ul>
+<li>could be pSB1C3-I13500 without R0011</li>
+</ul>
+<p class="obj">Objective: Insert mCherry G-block into pSB1C3-R0011-I13500</p>
+<p>PCR of mCherry G-block</p>
+<ul>
+<li>Q5 polymerase with Oligos SB1C3-dn and pDGC3-up</li>
+<li>4 tubes with 0, 0.4, 0.7, 1.0 uL template at 1 ng/uL</li>
+<li>45 sec extension, 66C anneal temp</li>
+</ul>
+<p>Agarose gel of PCR</p>
+<ul>
+<li>3 tubes look successful</li>
+</ul>
+<p>PCR cleanup of mCherry G-block PCR</p>
+<ul>
+<li>Qiagen kit</li>
+<li>40 uL at 27.6 ng/uL</li>
+</ul>
+<p class="obj">Objective: Various ligations</p>
+<p>Gel extraction and cleanup </p>
+<ul>
+<li>Each extraction included 2 cleaned digests combined into one lane (~100 uL)</li>
+<li>Cut and cleaned with Zymoclean prep kit</li>
+<li>pSB1C3-Csy4-scaffold cut with XbaI/PstI on 7/25</li>
+</ul>
+<ul>
+<li>pSB1C3 backbone 47.5 ng/uL in 30 uL</li>
+<li>Csy4-scaffold insert 15.4 ng/uL in 30 uL</li>
+</ul>
+<ul>
+<li>pSB1C3-Repeat-seq scaffold cut with XbaI/PstI on 7/25</li>
+</ul>
+<ul>
+<li>pSB1C3 backbone 74.8 ng/uL in 30 uL</li>
+<li>Repeat-seq scaffold insert 13.1 ng/uL in 30 uL</li>
+</ul>
+<ul>
+<li>pSB1C3-R0040-anti-tracrRNA cut with XbaI/PstI on 7/25</li>
+</ul>
+<ul>
+<li>pSB1C3 backbone 164.0 ng/uL in 30 uL</li>
+<li>R0040-anti-tracrRNA 176.0 ng/uL in 30 uL</li>
+</ul>
+<p>PCR Cleanup on gel extraction cleanups</p>
+<ul>
+<li>to &ldquo;superclean&rdquo;</li>
+<li>pSB1C3 4 tubes combined into 1: 469.6 ng/uL in 20 uL</li>
+<li>Repeat-seq scaffold: 23.9 ng/uL</li>
+</ul>
+<ul>
+<li>rough graph, probably not useful</li>
+</ul>
+<ul>
+<li>Csy4 scaffold: 16 ng/uL</li>
+</ul>
+<ul>
+<li>rough graph, probably not useful</li>
+</ul>
+<ul>
+<li>R0040-anti-tracrRNA: 7.4 ng/uL</li>
+</ul>
+<ul>
+<li>rough graph, probably not useful</li>
+</ul>
+<p class="obj">Objective: Measurement interlab study</p>
+<p>CIP treatment of backbones</p>
+<ul>
+<li>Using Charlie&rsquo;s CIP stock</li>
+<li>pSB1C3-K823005 cut with SpeI/PstI on 7/25 (2 tubes)</li>
+<li>pSB1C3-K823012 cut with SpeI/PstI on 7/25 (2 tubes)</li>
+</ul>
+<p>Gel extraction and cleanup</p>
+<ul>
+<li>2 cleaned digests combined into one lane (~100 uL)</li>
+<li>Cut and cleaned with Zymoclean prep kit</li>
+<li>pSB1C3-E0240 cut with XbaI/PstI on 7/25</li>
+</ul>
+<ul>
+<li>pSB1C3 backbone 116.4 ng/uL in 30 uL</li>
+<li>E0240 86.5 ng/uL in 30 uL</li>
+</ul>
+<p>PCR Cleanup of CIP treatment and gel extraction cleanup</p>
+<ul>
+<li>Qiagen kit</li>
+<li>pSB1C3-K823005 SpeI/PstI CIP treated: 119.8, 119.7 ng/uL</li>
+<li>pSB1C3-K823012 SpeI/PstI CIP treated: 60.9, 62.3 ng/uL</li>
+<li>E0240 XbaI/PstI extracted: 56.5 ng/uL in 20 uL (&ldquo;superclean&rdquo;)</li>
+</ul>
+<p>Ligations</p>
+<ul>
+<li>E0240 into two backbones: pSB1C3-K823005 and pSB1C3-K823012</li>
+<li>pSB1C3-K823005 SpeI/PstI/CIP: 100 ng = 0.87 uL</li>
+<li>pSB1C3-K823012 SpeI/PstI/CIP: 100 ng = 1.67 uL</li>
+<li>E0240 XbaI/PstI: 300 ng = 6 uL</li>
+<li>2 BO controls and 1 IO control</li>
+<li>20 mins at rt</li>
+<li>Heat shock transformation and plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Test crRNA repression of GFP reporter</p>
+<p>Dilute cultures to grow in log phase for flow</p>
+<ul>
+<li>1/1000 dilution into 50 mL</li>
+<li>18 samples: 3 copies each of 6 strains (labelled 2-19)</li>
+<li>Samples 2-4: DH5alpha-Z1 (background) </li>
+</ul>
+<ul>
+<li>grown in LB+Spec</li>
+</ul>
+<ul>
+<li>Samples 5-7: DH5alpha-Z1 + pSB6A1-K608012 (reporter positive control)</li>
+</ul>
+<ul>
+<li>grown in LB+Amp</li>
+</ul>
+<ul>
+<li>Samples 8-10: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9 (non-target control)</li>
+</ul>
+<ul>
+<li>grown in LB+Amp+Cm</li>
+</ul>
+<ul>
+<li>Samples 11-13: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP1</li>
+</ul>
+<ul>
+<li>grown in LB+Amp+Cm</li>
+</ul>
+<ul>
+<li>Samples 14-16: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP2</li>
+</ul>
+<ul>
+<li>grown in LB+Amp+Cm</li>
+</ul>
+<ul>
+<li>Samples 17-19: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP3</li>
+</ul>
+<ul>
+<li>grown in LB+Amp+Cm</li>
+</ul>
+<p>Flow cytometry </p>
+<ul>
+<li>Samples grown for 4.5 hrs after initial dilution</li>
+<li>1/50 final dilution of sample into PBS, placed on ice until run</li>
+<li>parameters (same as previous GFP runs):</li>
+</ul>
+<ul>
+<li>FSC 350V log3</li>
+<li>SSC 350V log3</li>
+<li>B1 350V log5</li>
+<li>Trigger 10</li>
+<li>10,000 event limit (100,000 events used in the past)</li>
+</ul>
+<ul>
+<li>Results</li>
+</ul>
+<ul>
+<li>~3-fold repression of reporter with crRNA_GFP1 or crRNA_GFP3</li>
+<li>crRNA_GFP2 did not repress</li>
+<li>non-target control repressed little or not at all</li>
+<li>See document &ldquo;<a href="https://docs.google.com/file/d/0B8nzM6b_weNTaDZwLWtqc0FPcG8/edit">Flow stats 7-28-14</a>&rdquo; for data details</li>
+</ul>
+<!--https://2014.igem.org/File:7-28-14-flow.png or https://static.igem.org/mediawiki/2014/f/f2/7-28-14-flow.png -->
+<a href="https://static.igem.org/mediawiki/2014/f/f2/7-28-14-flow.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/f2/7-28-14-flow.png"></a>
+</div>
+</div>
+<div class="day">
+<a id="jul29"><h2>July 29</h2></a>
+<div class="lab">
+<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p>
+<p>Minipreps</p>
+<ul>
+<li>pSB4K5-J04450 (4 copies)</li>
+<li>pSB1C3-R0040-anti-tracrRNA (4 copies)</li>
+</ul>
+<p>Prep-scale digest</p>
+<ul>
+<li>pSB4K5-J04450 (4 copies) with EcoRI/SpeI</li>
+<li>pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI</li>
+</ul>
+<p>Gel extraction and cleanup of digests</p>
+<ul>
+<li>Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450</li>
+</ul>
+<ul>
+<li>2 digests per gel well</li>
+<li>2.04 g total gel eluted into two tubes:</li>
+</ul>
+<ul>
+<li>51.4 ng/uL and 94.5 ng/uL, 30 uL each</li>
+</ul>
+<ul>
+<li>Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA</li>
+</ul>
+<ul>
+<li>2 digests per gel well</li>
+<li>1.61 g total gel eluted into two tubes:</li>
+</ul>
+<ul>
+<li>99.4 ng/uL and 82.3 ng/uL, 30 uL each</li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Colony PCR</p>
+<ul>
+<li>24 colonies of potential pSB1C3-R0011-I13500</li>
+</ul>
+<ul>
+<li>8 colonies from 7/15 ligation</li><li>4x4=16 colonies from 4 different 7/21 ligation plates</li>
+</ul>
+<ul>
+<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
+<li>extension time 45 sec, anneal temp 62C</li>
+</ul>
+<p>Agarose gel of colony PCR results</p>
+<ul>
+<li>colony #19 is only promising result</li>
+</ul>
+<p>Analytical digest</p>
+<ul>
+<li>pSB1C3-R0011 copies 2, 4</li>
+<li>pSB1C3-I13500 copies 2, 4</li>
+<li>with MfeI/NcoI</li>
+<li>To test whether enzymes or original parts are the source of ligation difficulties</li>
+</ul>
+<p>Agarose gel of digest results</p>
+<ul>
+<li>Order:</li>
+</ul>
+<ul>
+<li>1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)</li>
+<li>2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)</li>
+<li>3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)</li>
+<li>4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)</li>
+</ul>
+<ul>
+<li>Results:</li>
+</ul>
+<ul>
+<li>R0011 both copies only show one band</li>
+<li>I13500 both copies appear correct</li>
+</ul>
+<p>Cultured colonies</p>
+<ul>
+<li>Potential pSB1C3-R0011-I13500 #19 from colony PCR</li>
+<li>pSB1C3-R0011 (3 copies) from original transformation </li>
+</ul>
+<ul>
+<li>suspicion that our R0011 is incorrect--could be just bad copies</li>
+</ul>
+<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p>
+<p>Culture colonies</p>
+<ul>
+<li>pSB1C3-R0040 (4 copies)</li>
+</ul>
+<p class="obj">Objective: Measurement interlab study</p>
+<p>Plate results</p>
+<ul>
+<li>both experimental and both backbone-only plates all had hundreds of colonies</li>
+<li>insert-only controls had no colonies</li>
+</ul>
+<p>Colony PCR</p>
+<ul>
+<li>8 colonies of potential pSB1C3-K823005-E0240</li>
+<li>8 colonies of potential pSB1C3-K823012-E0240</li>
+<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
+<li>extension time 45 sec, anneal temp 62C</li>
+</ul>
+<p>Agarose gel of colony PCR results</p>
+<ul>
+<li>1-8: pSB1C3-K823005-E0240</li>
+<li>9-16: pSB1C3-K823012-E0240</li>
+<li>Results: no successes</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul30"><h2>July 30</h2></a>
+<div class="lab">
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Minipreps</p>
+<ul>
+<li>pSB1C3-R0011-I13500 #19</li>
+</ul>
+<ul>
+<li>concentration 246.4 ng/uL</li>
+</ul>
+<ul>
+<li>pSB1C3-R0011 (3 copies)</li>
+</ul>
+<ul>
+<li>concentrations 187.6, 178.8, 212.8 ng/uL</li>
+</ul>
+<p>Analytical digest</p>
+<ul>
+<li>pSB1C3-R0011-I13500 #19</li>
+</ul>
+<ul>
+<li>three digests in cutsmart, 10 uL final volume each</li>
+</ul>
+<ul>
+<li>NcoI</li>
+<li>MfeI</li>
+<li>XbaI/PstI</li>
+</ul>
+<ul>
+<li>pSB1C3-R0011 (3 copies)</li>
+</ul>
+<ul>
+<li>with NcoI/MfeI in cutsmart, 10 uL final volume</li>
+</ul>
+<p>Agarose gel of digest results</p>
+<ul>
+<li>1. pSB1C3-R0011-I13500 #19 with NcoI</li>
+<li>2. pSB1C3-R0011-I13500 #19 with MfeI</li>
+<li>3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI</li>
+<li>4. pSB1C3-R0011 #1 with NcoI/MfeI</li>
+<li>5. pSB1C3-R0011 #2 with NcoI/MfeI</li>
+<li>6. pSB1C3-R0011 #3 with NcoI/MfeI</li>
+<li>Results: hard to explain</li>
+</ul>
+<ul>
+<li>R0011-I13500 not correct</li>
+<li>R0011 all three copies only cut once </li>
+</ul>
+<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5 </p>
+<p>Treat pSB4K5 with Antarctic Phosphatase</p>
+<ul>
+<li>2 copies digested on 7/29, combined into one tube with 100 uL final volume</li>
+<li>2 hrs at 37C</li>
+</ul>
+<p>PCR cleanup of AP-treated pSB4K5</p>
+<ul>
+<li>Qiagen kit</li>
+<li>concentration 45.2 ng/uL in 50 uL</li>
+</ul>
+<p>Ligation of pSB4K5 and R0040-anti-tracrRNA</p>
+<ul>
+<li>2 experimental plates with 3x and 6x molar insert</li>
+<li>pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL</li>
+<li>R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL</li>
+<li>Backbone-only and insert-only controls</li><li>Transformed via heat shock and plated on LB+G418</li>
+</ul>
+<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p>
+<p>Minipreps</p>
+<ul>
+<li>pSB1C3-R0040 (4 copies)</li>
+</ul>
+<ul>
+<li>concentrations 47.4, 80.7, 109.7, 193.5</li>
+</ul>
+<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
+<p>Treat pSB1C3 with Antarctic Phosphatase</p>
+<ul>
+<li>cleaned copy from 7/28 with 100 uL final volume</li>
+<li>2 hrs at 37C</li>
+</ul>
+<p>PCR cleanup of AP-treated pSB1C3</p>
+<ul>
+<li>Qiagen kit</li>
+<li>concentration 104.5 ng/uL in 50 uL</li>
+</ul>
+<p>Ligation of pSB1C3 and dCas9-tracrRNA</p>
+<ul>
+<li>2 experimental plates</li>
+</ul>
+<ul>
+<li>3x molar dCas9-tracrRNA &ldquo;insert&rdquo;</li>
+</ul>
+<ul>
+<li>pSB1C3 100 ng = 1 uL</li>
+<li>dCas9-tracrRNA 600 ng = 7.5 uL</li>
+</ul>
+<ul>
+<li>3x molar pSB1C3 &ldquo;backbone&rdquo;</li>
+</ul>
+<ul>
+<li>pSB1C3 150 ng = 1.5 uL</li>
+<li>dCas9-tracrRNA 100 ng = 1.2 uL</li>
+</ul>
+<ul>
+<li>Backbone-only and insert-only controls</li>
+<li>Transformed via heat shock and plated on LB+Cm</li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jul31"><h2>July 31 </h2></a>
+<div class="lab">
+<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p>
+<p>Plate results:</p>
+<ul>
+<li>Hundreds of colonies on both experimental plates</li><li>20-30 colonies on backbone-only control</li>
+<li>0 colonies on insert-only control</li>
+</ul>
+<p> Cultured colonies</p>
+<ul>
+<li>4 copies of pSB4K5-R0040-anti-tracrRNA</li>
+</ul>
+<p class=”obj”>Objective: Switch dCas9-tracrRNA and dCas9-tracrRNA-crRNA into pSB1C3</p>
+<p>Plate results:</p>
+<ul>
+<li>Experimental plate 2 (higher pSB1C3 concentration) had 2 colonies</li>
+<li>Experimental plate 1 had no colonies</li>
+<li>Backbone-only control had 1 colony, insert-only control had 0</li>
+</ul>
+<p>Cultured colonies</p>
+<ul>
+<li>2 copies of potential pSB1C3-dCas9-tracrRNA</li>
+</ul>
+<p>PCRs of tracrRNA-dCas9 and tracrRNA-dCas9-crRNA</p>
+<ul>
+<li>8 tubes of each, with 1 uL 1 ng/uL pdCas9 template per tube</li>
+<li>tracrRNA-dCas9 oligos: dCas9tracr-up and dCas9tracr-dn</li>
+<li>tracrRNA-dCas9-crRNA oligos: dCas9tracr-up and pdCas9-dn</li>
+<li>Q5 polymerase</li>
+<li>64C anneal temp, 2:30 extension time</li>
+</ul>
+<p>Agarose gel of PCRs</p>
+<ul>
+<li>1-8: tracrRNA-dCas9-crRNA</li>
+<li>9-16: tracrRNA-dCas9</li>
+<li>Results: all 16 lanes look good</li>
+</ul>
+<p>PCR cleanup of PCRs</p>
+<ul>
+<li>8 tubes into 50 uL for each of two PCRs</li>
+<li>Concentrations</li>
+</ul>
+<ul>
+<li>tracrRNA-dCas9-crRNA: 350 ng/uL</li>
+<li>tracrRNA-dCas9: 335 ng/uL</li>
+</ul>
+<p>Prep-scale digest of 2 PCRs</p>
+<ul>
+<li>tracrRNA-dCas9-crRNA and tracrRNA-dCas9</li>
+<li>Each in 100 uL final volume with 2 uL each of XbaI/PstI/DpnI</li>
+</ul>
+<p>PCR cleanup of digests</p>
+<ul><li>Into 50 uL final volume </li></ul>
+<p class="obj">Objective: Put R0040 in front of Csy4 scaffold and Repeat-seq scaffold</p>
+<p>Prep-scale digest</p>
+<ul>
+<li>pSB1C3-R0040 (2 copies) with 2.5 uL each of SpeI/PstI</li>
+<li>100 uL final volume each copy</li>
+</ul>
+<p>PCR cleanup of digests</p>
+<ul><li>Into 50 uL final volume</li></ul>
+<p class="obj">Objective: Triple-transformation of R0040-anti-tracrRNA into Z1-reporter-crRNA_GFP cells</p>
+<p>Cultured cells in morning</p>
+<ul>
+<li>straight from frozen stocks into 2 mL LB+Cm+Amp</li>
+<li>DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP1</li>
+<li>DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP3</li>
+</ul>
+<p>Cultured cells in evening</p>
+<ul>
+<li>1 mL from morning culture into 50 mL flask for overnight growth</li>
+<li>To make chemically competent cells</li>
+</ul>
+<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
+<p>Cultured colonies</p>
+<ul>
+<li>2 copies of pSB1C3-R0011 from streak plate of original frozen stock</li>
+</ul>
+<!--
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+</div>
+</div>
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+<!--
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+-->
+<!--
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