Team:Duke/Notebook/MayJun

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Latest revision as of 15:10, 16 October 2014

May 29

Objective: Prepare Chemically competent E. Coli

Matthew Faw

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

Transformation

June 1

Objective: Transform Chemically competent E. Coli (CCEC)

Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

Followed Charlie’s Cloning Protocols

Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful

Plated the transformed DNA and put in incubator at 37C
Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

Look at plates, compare cultures

June 2

Objective: Redo yesterday’s transformation

Matthew Faw

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

Followed Charlie’s Cloning Protocols

Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure

Plated the transformed DNA on 6 separate plates, put in 37C overnight
Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

Look at plates, compare cultures, etc.

June 4

Objective: Prepare buffers and mediums for new CCEC protocol

Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper

Prepare SOB Medium for bacterial transformation

Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
Made 1 L, autoclaved and stored in two 500 mL containers in cold room

medium still appeared cloudy before autoclave--may just be new recipe

Prepare CCMB 80 Buffer for making chemically competent E. coli cells

Protocol from iGEM’s parts website
Made 1 L, filtered and stored in two 500 mL containers in cold room

pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed

Autoclave two 500 mL culture flasks

For CCEC protocol
With water inside to remove detergent residues

Next steps:

Prepare CCEC

Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC

Matthew Faw

The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates.

Followed Charlie’s Cloning protocols,with slight modifications

Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure

Plated the transformed DNA on 2 separate plates, put in put in 37C overnight
Results: No colonies grew (6/5/14)

Next Steps:

Examine plates to see if any cultures grew
Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here:
Lab currently in the process of making these CCEC

Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped

Matthew Faw, Charlie Cooper

Prepare pdCas9 and pCsy4 to be miniprepped tomorrow

Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol
Put pCsy4 in a culture tube with 5ml SOC+Amp
Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them

Next steps:

Miniprep plasmid DNA

June 5

Objective: Attempt to grow some transformed cell cultures with Charlie's CCEC.

Matt Faw, Charlie Cooper

Results: Plates transformed 6/4 had no colonies (see for results)

Objective: Glycerol stocks and Miniprep our plasmid DNA

Matt Farnitano, Matt Faw, TJ Ciesla

Stored glycerol stocks (15% glycerol) of pdCas9 (A1) and pdCas9 (A2) at -80

Miniprep our pdCas9 and pCsy4

We miniprepped both pdCas9 and pCsy4 following standard protocol included in the miniprep kit. We were unsure of how successful our miniprep of pdCas9 would be because it was low copy.
We took our own miniprep kit, prepared all solutions necessary, and labeled as ours. Use this miniprep kit from now on.
We analyzed the concentrations of our plasmids with the spectrophotometer.
Results (6/5/14): Both minipreps were successful, with pdCas9 at concentration 103.4 ng/ulpCsy4 at concentration 138.1ng/ul
Stored in iGEM 2014 Box 1, A1 and A2

Next steps:

Wait for oligos ordered 6/4/14 in order to use these plasmids in construction.

June 6

Objective: Observe June 5 plates

Matt Faw

Results: the colonies that we left to grow in 2mL of SOB medium in the shaker at 37C didn’t grow…. Not sure if there’s a problem with the SOB medium, the cells, etc. Today we’ll try growing various bacteria in the SOB medium to try to figure out what went wrong

Objective: Perform PCR on the pdCas9 template using oligos 5/18 and 6/18 that arrived today to obtain the desired biobrick sequence

Matthew Faw, TJ Ciesla, Charlie Cooper

PCR

Followed standard PCR procedure on the pdCas9

0.5 uL Phusion HF polymerase/reaction
0.25 uL each oligo
1 uL dNTP mix

Oligo 5: 5p-EcoR1-Xbal-Rpt
Oligo 6: Spe1-Repeat-3p

Note: Created new box called iGEM Cloning Project. dNTP’s in A1, and EcoR1-Xbal-Rptg-Rpt-Spe1-PCR in A2

PCR was successful, with concentration 115.4 ng/ul. Pooled 3x50uL reactions with PCR cleanup protocol and eluted in to 40uL dH2O

Next steps:

Digest and transformation for biobrick submission

June 9

Objective: Grow and confirm two backbone plasmids with “RFP coding device” insert from iGEM registry plates

Matthew Farnitano and TJ Ciesla

Miniprepped and stored glycerol stocks of two copies each of pSB6A1-BBa_J04450 and pSB1C3-BBa_J04450

Glycerol stocks in iGEM bacteria box 1, A3 and A4.
Followed Qiagen kit for miniprep procedure
All four E. coli strains visibly reddish before miniprep (sign that RFP is expressed)
Results: All four successful, concentrations (ng/uL):

6A1-1: 429.2, 6A1-2: 461.2,1C3-1: 337.2, 1C3-2: 294.2

10 lanes:

1. BBa_J04450+pSB6A1-1(uncut)
2. BBa_J04450+pSB1C3-1 (uncut)
3. A-1 cut with Kpn1-expected 4.8 and 0.25 kb bands
4. A-2 cut with Kpn1-expected 4.8 and 0.25 kb bands
5. A-1 cut with Afe1-expected 3.8, 0.9, and 0.28 bands
6. A-2 cut with Afe1-expected 3.8, 0.9, and 0.28 bands
7. C-1 cut with Kpn1-expected 2.9, 0.25 bands
8. C-2 cut with Kpn1-expected 2.9, 0.25 bands
9. C-1 cut with Nco1-expected 1.7, 1.4 bands
10. C-2 cut with Nco1-expected 1.7, 1.4 bands

Results: All expected bands are present in all samples. A faint unexpected band is present at ~2.5-3.0 kb in lanes 3, 4, 5, 6, maybe 7 and 8. Band is not strong enough to be a full piece of the plasmid

Gel picture:

Note: Kept copies 6A1-2 (glycerol box A-3) and 1C3-1 (glycerol box A-4), discarded others. TJ Ciesla and Charlie Cooper streaked a plate from 1C3-1 for use later

Objective: Grow and confirm two backbone plasmids with “RFP coding device” insert from iGEM registry plates

Matthew Farnitano and TJ Ciesla

Ligation-scale restriction digest of pSB1C3-BBa_J04450 and 6/6/14 PCR product

EcoRI-HF and SpeI-HF (in Cutsmart buffer)
35 uL pSB1C3 DNA (337.2 ng/uL) in 50 uL reaction for 3 hrs
35 uL Repeat-gRNA-Repeat PCR product in 50 uL reaction for 3 hrs

Made 0.8% agarose TAE gel to separate pSB1C3 backbone from insert

Ran pSB1C3-BBa_J04450 digest on gel

Expected to see two bands, 2.0 kb and 1.1 kb.

Results: only one large band ~3.0 kb with faint bands slightly below

Gel photo:

June 10

Objective: Assemble Repeat-gRNA-Repeat scaffold in pSB1C3 backbone

Matthew Farnitano and TJ Ciesla

Analytical Restriction Digest

To test why digest 6/9/14 did not work as expected
pSB1C3-BBa_J04450 in three cuts, with pCC415 as a control cut (see gel results below)

Agarose gel of restriction digest

1. pCC415 cut with SacI-HF: expecting two bands
2. pSB1C3-BBa_J04450 cut with SacI-HF and SpeI-HF: expecting 2.0 and 1.1 kb bands
3. pSB1C3-BBa_J04450 cut with SacI-HF and EcoRI-HF: expecting 2.3 and 0.9 kb bands
4. pSB1C3-BBa_J04450 cut with SpeI-HF and EcoRI-HF: expecting 2.0 and 1.1 kb bands

Results: Expectations met

Gel photo:

Isolate DNA from restriction digest of PCR product (Repeat-seq-Repeat)

DNA isolated using Qiagen Spin columns - last few steps of miniprep protocol
DNA is 75 ng/µL in 30 µL EB buffer

Objective: Grow plasmids

Grow bacteria in medium

1. DH5α (Charlie’s) in SOC for future transformation
2. DH5α, plate 3, 15B in LB+CM* (pSB1C3-BBa_J06702)
3. DH5α, plate 2, 6F in LB+CM* (pSB1C3-BBa_R0040)
4. DH5α, plate 2, 17M in LB + CM* (pSB1C3-BBa_K741002)
5. DH5α, pSB1C3-BBa-J04450 in LB + CM**
6. DH5α, pSB6A1-BBa-J04450 in LB + amp**
*2, 3, 4 contain plasmids w/biobricks
**5 and 6 will be used to calibrate flow cytometry in 6/11/14

Next steps:

Miniprep, digest to confirm plasmids from 2, 3, 4

J06702 contains RBS-mCherry-T, to be PCRed out
R0040 contains pTet, to be PCRed out
K741002 contains pLac-RBS-GFP, and Terminator, both to be PCRed out

Dilute 5 and 6, use for flow calibration

June 11

Objective: Prepare chemically competent E. coli (DH5α)

Froze 6 1mL seed stocks of DH5alphas in 15% glycerol

From 6/10 culture
For use in iGEM Chemically Competent Cell protocol

Grow 2 seed stocks in SOC overnight for CCEC protocol

Each stock 1mL DH5alpha in 250mL SOC
In shaker at room temperature (started 6:30pm)

Next Steps:

Complete protocol with cultures

Objective: Prepare biobrick plasmids for cloning

Froze 15% glycerol stocks of three biobrick plasmids at -80C

DH5alpha, pSB1C3-BBa_K741002, stock location Box 1-A5
DH5alpha, pSB1C3-BBa_J06702, stock location Box 1-A6
DH5alpha, pSB1C3-BBa_R0040, stock location Box 1-A7

Miniprepped three plasmids

Standard Qiagen kit protocol
Results:

1C3-K741002 40uL at 98 ng/uL, iGEM box 1 A5
1C3-J06702 40uL at 114 ng/uL, iGEM box 1 A6
1C3-R0040 40uL at 170 ng/uL, iGEM box 1 A7

Analytical restriction digest of minipreps to confirm

Each of three plasmids in EcoRI-HF/SpeI-HF and in PstII-HF

All in cutsmart buffer, 2 hrs at 37C

Agarose gel of restriction digest

1. 1C3-K741002 in EcoRI-HF/SpeI-HF : expected 2.0, 1.1 kb bands
2. 1C3-K741002 in PvuII-HF : expected 1.9, 0.89, 0.4 kb bands
3. 1C3-J06702 in EcoRI-HF/SpeI-HF : expected 2.0, 0.89 kb bands
4. 1C3-J06702 in PvuII-HF : expected 2.0, 1.0 kb bands
5. 1C3-R0040 in EcoRI-HF/SpeI-HF : expected 2.0, 0.08 kb bands
6. 1C3-R0040 in PvuII-HF : expected 2.1 kb band

Results: Order of tubes in gel was not recorded. Lanes probably good, but should retest to be sure.

Gel photo:

Next steps:

Reconfirm plasmids
PCR pLac-RBS-GFP from K741002
PCR RBS-mCherry-Term from J06702
Isolate pSB1C3
SLIC to make pDGC3

Objective: Prepare biobrick plasmids for cloning

Diluted E. coli to grow to log phase for flow

DH5alpha (SOC), pSB1C3-BBa_J04450 (LB+Cm), and pSB6A1-BBaJ04450 (LB+Amp)
Each strain three dilutions: 1/100, 1/250, 1/500
Growing from ~10am
Note: Overnight culture of 1C3 had no visible red, but culture of 6A1 was strong reddish-pink. Possible that RFP is not expressed well in 1C3.

Flow Cytometry Parameter setting

1. calibration beads
2. messing around with parameters
3. pSB6A1-BBa_J04450 1/500 initial dilution
4. pSB1C3-BBa_J04450 1/500 initial dilution, 1/50 final dilution at flow time
5. DH5alpha 1/500 initial dilution, 1/50 final dilution at flow time
6. pSB6A1-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time
7. pSB1C3-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time

Notes: pSB1C3-BBa_J04450 showed strong, clear red fluorescence at both dilutions. pSB6A1 showed a bimodal fluorescence with some cells matching 1C3 fluorescence and others matching DH5alpha natural fluorescence. All samples showed two peaks of FS:SS data. These peaks did not correlate with the bimodal fluorescence seen in 6A1.

Parameters settled on (subject to change): FS: 350 volts, log 3 SS: 350 volts, log 3 Y2: 400 volts, log 5

Next Steps:

Perform formal data analysis

Objective: Create Csy4-gRNA-Csy4 Scaffold using G-block

G-block arrived today, CC split with Sargis

PCR of G-block to obtain iGEM piece

Used oligos pSB1C3-up and pSB1C3-down
iGEM protocol with 65C anneal, 20 sec extension

Agarose Gel of PCR product

Used HindIII ladder instead of 2-log
Results looked good, hard to tell exactly with ladder
Some junk in no template control, but absent in other tubes

Gel photo:

PCR cleanup of G-block PCR

Results: 30 uL product at 167.6 ng/uL

SLIC of pSB1C3 and Csy4-gRNA-Csy4 and Transformed

1 uL pSB1C3 from gel, purified (today), cut with EcoRI/SpeI
0.2 uL g-block in 10 uL reaction
One no-backbone control (not enough backbone for no-insert control
Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm

Objective: Create Repeat-seq-Repeat Scaffold

Gel clean-up of pSB1C3 cut from EcoRI/SpeI gel

Gel picture:

Digested and cut 6/10 and frozen overnight
Used Zymoclean prep kit
Results: Obtained 10 uL product at 133.1 ng/uL

Ligation of pSB1C3 with Repeat-seq-Repeat and Transform

4 uL pSB1C3 backbone from gel purified today
2 uL Repeat-seq-Repeat insert cut 6/9, cleaned 6/10
One no-insert control (next time do a no-backbone control as well)
Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm

June 12

Objective: Create Repeat-seq-Repeat construct and Csy4-gRNA-Csy4 construct

Results of transformation: no colonies on SLIC or Ligation plate

Controls also blank
Made new backbone for use (see below)

Overnight ligation of Repeat-seq-Repeat PCR with pSB1C3/p>

~100 ng backbone (2 uL) with ~30 ng insert (0.5 uL)

in 20 uL reaction

Two controls: one with no backbone, one with no insert
Left in heat block in cold room (16C) at 6pm for overnight ligation

Next steps:

Do a parallel room temp. ligation
SLIC of Csy4 construct
Transform all of the above, use Charlie’s CCEC until ours are confirmed

Objective: Obtain pSB1C3 backbone (various cloning uses)

Restriction digest of pSB1C3-K741002 and pSB1C3-J06702

25 uL each backbone in 100 uL reactions
SpeI-HF and EcoRI-HF in Cutsmart buffer (11am start)

Agarose gel of product for extraction

One lane looked as expected (2.0 and 0.8 kb bands)
Other lane had 2.0 kb backbone band but no insert band

possibly a faint band ~.1 kb

Extracted backbone (2.0 kb band) from both lanes separately

Gel photo:

Gel purification of pSB1C3

Used Zymoclean prep kit
Final elution:

Confirmed piece 20 uL at ng/uL (teal tube)
Unconfirmed piece 20 uL at ng/uL (orange tube)

Notes: it is possible that the lack of insert is due to mislabelled tubes. Analytical digests and reculturing of the three plasmids in pSB1C3 (K741002, J06702, R0040) will determine if this is the case and correct any mislabelling.

Objective: Create pDGC3

PCR of pLac-RBS-GFP from pSB1C3-K741002

With oligos pSB1C3-up and GFP-A-Z-3p
Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL)
iGEM protocol with 30 sec extension, 65C anneal

PCR of RBS-mCherry-Term from pSB1C3-J06702

With oligos 5p-Z-Asc1-RBS-mCherry
Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL)
iGEM protocol with 30 sec extension, 67C anneal

Agarose gel of PCR results

Lanes 1-4: pSB1C3-K741002 PCR

No product (fuzzy primer bands only)

Lanes 5-8: pSB1C3-J06702 PCR

Some product present at low concentrations

Gel photo:

PCR cleanup of pSB1C3-J06702 PCR

To see if low concentrations are enough to work with

<50 ng/uL present, need to redo PCR

Next steps: Redo PCR (once proper plasmids are confirmed), confirm, cleanup, SLIC

Objective: Prepare Chemically Competent E. coli

At ~11am, OD600 absorption rate for the cultures was .127 and .137

Aiming for OD600 of 2-3
Let grow for 50 more minutes (approx. one doubling time)
OD600 still low (.163, .178), let grow another hour
OD600 >0.2

Completed protocol as stated from iGEM website

Used conical-bottom tubes instead of flat-bottom, resulting in difficulty of resuspending cell pellets. Had to vortex pellets substantially to resuspend, which may reduce competence.
50-ml tubes used were smaller than those used by iGEM headquarters. Result was more tubes, each with suggested volume, so final culture was too dilute (OD < .1). Pelleted and resuspended in half the volume
Final volume 50 uL (for a double batch), in 50 tubes
Tubes in -80C new box, “iGEM chemically competent cells”

Next steps: Test competence with transformation of known circular plasmid

Objective: Reconfirm plasmids to check for mislabelling

Streaked single plate with frozen stocks of three plasmids in pSB1C3

K741002, J06702, R0040
All on same LB+Cm plate, because it was the last one

Need to make more

Incubated overnight at 37C

Next steps:

Culture, miniprep and digest to confirm correct insert
Digest current miniprep DNA as well
Re-label tubes if necessary

June 13

Objective: Reconfirm plasmids in pSB1C3 backbone (possible mislabelling event)

Analytical Restriction Digest of 3 plasmids (2 digests each)

Each plasmid in PvuII-HF and in EcoRI-HF/SpeI-HF
All reactions in cutsmart buffer
Note that plasmid names are as labelled, might not be what is present

Agarose gel of results:

1. pSB1C3-BBa_K741002 tube in PvuII-HF

2. pSB1C3-BBa_J06702 tube in PvuII-HF

3. pSB1C3-BBa_R0040 tube in PvuII-HF

4. pSB1C3-BBa_K741002 tube in EcoRI-HF/SpeI-HF

5. pSB1C3-BBa_J06702 tube in EcoRI-HF/SpeI-HF

6. pSB1C3-BBa_R0040 tube in EcoRI-HF/SpeI-HF

Results: No DNA in all K74 and J06 tubes. Tubes labelled R0040 appear to match expected for J06702

K74 and J06 were 1 ng/uL dilutions, so DNA was likely too dilute to show up

Hypothesis: R0040 and J06702 tubes are flipped. Possible that K741002 tube is incorrect as well
gel picture:

Objective: Create pDGC3 using biobrick parts

PCR of pLac-RBS-GFP from BBa_K741002

8 tubes, with two templates in 4 tubes each (0, .4, .7, 1.0 uL template)

“K” template is tube labelled as BBa_K741002 (1 ng/uL dilution)
“J” tube is tube labelled as BBa_J06702 (1 ng/uL dilution)

Trying in case this tube is mislabelled and contains K74

Oligos pSB1C3up and GFP-A-Z-3p
iGEM protocol with 30 sec extension time and 65C anneal

PCR of RBS-mCherry-Term from BBa_J06702

4 tubes at 0, .4, .7, and 1.0 uL template

Used tube labelled “R” as template

Suspect that this tube is mislabelled and contains J06

Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3-down
iGEM protocol with 30 sec extension time and 67C anneal

Agarose gel of PCRs

Lanes 1-4: K741002 PCR with “K” template

Lanes 5-8: K741002 PCR with “J” template

Lanes 9-12: J06702 PCR with “R” template

Results: primer bands only on all K741002 lanes. Strong expected band in lanes 10-12 indicating succesful PCR of J06702

Further evidence that “R” is mislabelled and contains “J”

gel picture:

PCR cleanup of J06702 PCR

Objective: Remove BbsI site from mCherry in J06702

Restriction digest of tube labelled R0040

Hopefully tube contains J06702 as predicted earlier
With Bbs1 in buffer 2.1 with BSA

50 uL reaction with 25 uL DNA

Agarose gel of restriction digest

Results: Looks as expected, hard to tell with single band
gel photo:

PCR cleanup of Restriction digest

Eluted in 30 uL
Still need to check concentration
Frozen at -20C

Objective: Create Repeat-seq-Repeat construct in pSB1C3

Ligation of Repeat-seq-Repeat PCR to pSB1C3 (confirmed green tube)

Three identical tubes: “controls” actually have all pieces in them
20 uL reactions with 2 uL (100 ng) backbone and 0.5 uL (30 ng) insert
Room temp for 1 hour

Transformation of ligations from overnight (6/12) and rt (6/13)

6 total tubes: 4 experimental and 2 controls (both from overnight)

Objective: Reconfirm and miniprep from frozen stocks of 3 plasmids in pSB1C3

Cultured 2 colonies each from 3 plasmid strains on 6/12/14 plate

pSB1C3-K741002, pSB1C3-J06702, and pSB1C3-R0040

TODO for weekend:

Miniprep cultures from frozen stock reconfirmations (Sat)
Pick and culture colonies from 6 ligation plates (Sat)
Miniprep cultures from ligation plates (Sun)

June 15

Charlie Cooper

Gblock SLIC in to pSB1C3 conducted by CBC

Objective: gRNA scaffold for 1) submission as a biobrick and 2) use for generating Csy4/ gRNA arrays

	backbone + insert	bb only	io
digested & purified backbone at ~50ng/uL	2	2	0
PCRd gBlock	1	0	1
buffer 2	1	1	1
10x BSA	1	1	1
water	5	6	7

all but enzyme pre-mixed in 500uL eppies. enzyme added and incubate 2.5m at RT before icing tubes for 10m. 65 uL CBC’s chem. comp DH5alpha then 20m on ice, 45s 42C shock, 2m on ice, 60m recovery on SOC then spread on LB+Cm overnight.
Results: no colonies the next day on any plates.

SLIC repair of BbsI cut site in mCherry by CBC

Objective: BbsI/golden gate compatible mCherry for gRNA or crRNA array assembly schemes

1 uL each of mCherry-Bbs1-left and mCherry-Bbs1-right at 100uM added to 500uL eppies and evaporated in SpeedVac for backbone + insert and insert-only control. placed on ice to pre-cool

	backbone + insert	bb only	io
bbsi-dig backbone (~5 0ng/uL)	2	2	0
oligos (evapd)	2	0	2
buffer 2	1	1	1
10x BSA	1	1	1
water	6	6	8

backbone + insert and bb only reactions treated with T4 poly as one 2x volume, then at 2.5m at RT 10 uL transferred to pre-chilled backbone + insert tube with waiting oligos. insert only reaction run as DNA-free dummy reaction for 2.5m in enzyme then transferred to pre-chilled tube with oligos.
65 uL chemically competent DH5alpha (from cbc) added at 10m on ice, incubated 20m, then heat shocked 45m at 42C then 2m on ice again before recovering for 60m in SOC and spread on plates.
Results: > 100 colonies on backbone-only control. improper spreading of backbone + insert makes it hard to tell, but there may be slightly more colonies there than on the bb-only.

June 16

Objective: Confirm 3 plasmids in pSB1C3 from frozen stocks.

Analytical restriction digest and agarose gel of plasmids

pSB1C3-BBa_K741002-1 in EcoRI-HF/SpeI-HF (expected 2.0, 1.0 kb bands)
pSB1C3-BBa_K741002-2 in EcoRI-HF/SpeI-HF (expected 2.0, 1.0 kb bands)
pSB1C3-BBa_J06702-1 in EcoRI-HF/SpeI-HF (expected 2.0, 0.9 kb bands)
pSB1C3-BBa_J06702-2 in EcoRI-HF/SpeI-HF (expected 2.0, 0.9 kb bands)
pSB1C3-BBa_R0040-1 in EcoRI-HF/SpeI-HF (expected 2.0, 0.1 kb bands)
pSB1C3-BBa_R0040-2 in EcoRI-HF/SpeI-HF (expected 2.0, 0.1 kb bands)
pSB1C3-BBa_K741002-1 in PvuII-HF (expected 1.8, 0.8, 0.4 kb bands)
pSB1C3-BBa_K741002-2 in PvuII-HF (expected 1.8, 0.8, 0.4 kb bands)
pSB1C3-BBa_J06702-1 in PvuII-HF (expected 2.0, 1.0 kb bands)
pSB1C3-BBa_J06702-2 in PvuII-HF (expected 2.0, 1.0 kb bands)
pSB1C3-BBa_R0040-1 in PvuII-HF (expected 2.1 kb band)
pSB1C3-BBa_R0040-2 in PvuII-HF (expected 2.1 kb band)

Results: All lanes look as expected. Confirmation that stocks are correct.

Previous minipreps and other tubes resulting from unconfirmed versions of these plasmids have been thrown out. The one exception is the successful PCR of RBS-mCherry-Term from BBa_J06702

Gel picture:

Objective: Obtain pSB1C3 backbone for various transformations

Prep-scale digest of pSB1C3-BBa_K741002

Using XbaI and SpeI-HF in cutsmart buffer
45 uL template in 100 uL reaction

Prep-scale digest of pSB1C3-Bba_R0040

Using EcoRI-HF and SpeI-HF in cutsmart buffer
45 uL template in 100 uL reaction

Gel extraction of digests to obtain pSB1C3 backbone

Cut top band from K741002(left on picture)

240mg gel, eluted in 20uL H2O

Cut only visible band from R0040(right on picture)

Cleaned in 2 tubes(200mg each), eluted in 10+10=20uL H2O

Cleaned up using Zymoclean prep kit

Used 300uL wash buffer instead of 200uL for extra clean

Gel picture:

Objective: Remove BbsI site from mCherry

SLIC transformations from 6/15(Charlie) showed colonies on the experimental plate as well as on the no-insert control

Cultured 6 colonies from plate for miniprep

Each in 5mL LB+Cm at 37C
In hopes of getting at least one mutated sequence

Cut BBa_J06702 with BbsI to try assembly again

4-hour digest with BbsI in NEBuffer 2.1 and BSA
45uL template in 100uL reaction
Gel picture:

PCR cleanup of BbsI cut BBa_J06702

Qiagen kit
Final concentration 83.9 ng/uL

Objective: Create pDGC3 construct

PCR of plac-RBS-GFP-A-Z from BBa_K741002

Used fresh Q5 polymerase and buffer
template pSB1C3-BBa_K741002 diluted to 1 ng/uL
4 tubes with 0, 0.4, 0.7, and 1.0 template
Oligos pSB1C3-up and GFP-A-Z-3p
iGEM protocol, extension time 30 sec, 65C anneal temp

Agarose gel of PCR product

Results: PCR appears to have worked (expected band size 1.0 kb)
Gel picture:

PCR cleanup

Qiagen kit
Final concentration 161.1 ng/uL in 30 uL

TODO:

SLIC of pSB1C3 SpeI/XbaI cut with G-block
SLIC of pSB1C3 J06702 BbsI cut with oligos for mutation
SLIC of pSB1C3-SpeI/XbaI cut with GFP PCR and mCherry PCR
Ligation of pSB1C3 SpeI/EcoRI cut with Repeat-seq-Repeat PCR
Possibly try Gibson of G-block assembly as well
Possibly transform biobrick 4-4G (Bba_B0030 in pSB1C3)

As a way to get pSB1C3 without gel extractions

Miniprep cultures of mCherry possible mutants

Cut with BbsI and another enzyme to test for successful mutants

June 17

Objective: Miniprep and test cultures resulting from attempted removal of BbsI from mCherry

Miniprep six colonies containing pSB1C3-BBa_J06702 with possible ∆BbsI

Qiagen kit
Concentrations (1-6): 284.5, 257.8, 268.2, 255.9, 242.2, 257.1 ng/uL

Analytical restriction digest and agarose gel of six possible ∆BbsI mutants

pSB1C3-J06702 (nonmutant control) cut with EcoRI-HF
pSB1C3-J06702 (nonmutant control) cut with BbsI/EcoRI-HF
pSB1C3-J06702 B1 cut with EcoRI-HF
pSB1C3-J06702 B1 cut with BbsI/EcoRI-HF
pSB1C3-J06702 B2 cut with EcoRI-HF
pSB1C3-J06702 B2 cut with BbsI/EcoRI-HF
pSB1C3-J06702 B3 cut with EcoRI-HF
pSB1C3-J06702 B3 cut with BbsI/EcoRI-HF
pSB1C3-J06702 B4 cut with EcoRI-HF
pSB1C3-J06702 B4 cut with BbsI/EcoRI-HF
pSB1C3-J06702 B5 cut with EcoRI-HF
pSB1C3-J06702 B5 cut with BbsI/EcoRI-HF
pSB1C3-J06702 B6 cut with EcoRI-HF
pSB1C3-J06702 B6 cut with BbsI/EcoRI-HF

Expected single band at 3.0 kb in EcoRI cuts
Successful mutants expected BbsI/EcoRI cuts to match EcoRI-only cuts
Unsuccessful nonmutants expected 2.5, 0.5 kb bands in BbsI/EcoRI cuts
Results: All six colonies unsuccessful (two bands in BbsI/EcoRI cut)

All controls looked as expected

gel picture:

Objective: Create various plasmids by Ligation/SLIC

Ligation to create pSB1C3-Repeat-seq scaffold

100 ng pSB1C3 cut with EcoRI/SpeI = 0.4 uL @ 30 ng/uL
30 ng Repeat-seq PCR = 0.4 uL @ 77 ng/uL
No-backbone and no-insert controls

SLIC to create pSB1C3-Csy4-gRNA-scaffold

100 ng pSB1C3 cut with SpeI/XbaI = 1.1 uL @ 91 ng/uL
30 ng (3xmolar) G-block PCR = 0.2 uL @ 167 ng/uL
No-backbone and no-insert controls

SLIC to create pDGC3 (with GFP and mCherry in pSB1C3)

100 ng pSB1C3 cut with SpeI/XbaI = 1.1 uL @ 91 ng/uL
150 ng (3xmolar) GFP-A-Z PCR = 2.5 uL @ 61 ng/uL
150 ng (3xmolar) mCherry PCR = 1.1 uL @ 132.8 ng/uL
No-insert control (no-backbone shared with Csy4 SLIC)

SLIC to remove BbsI from pSB1C3-J06702

100 ng pSB1C3-J06702 cut with BbsI = 1.2 uL @ 83.9 ng/uL
Oligos mCherry-BbsI-left and mCherry-BbsI-right (0.5 uL each)
No insert and no-backbone controls

Notes:

SLICs may have been left on ice too long (>10 mins) before cells were added

Large number of tubes for pipetting made timing uneven

All 11 samples plated on LB+Cm plates

Objective: Test CCEC from 6/12/14

Transformed uncut plasmid (pSB1C3-J06702-B5) with new CCEC

4 different concentrations (1/10 dilutions each):

200 ng, 20 ng, 2 ng, and 0.2 ng
Each in 10 uL tubes

Plated on LB+Cm

June 18

Objective: Make LB+Cm agar plates

Charlie’s plate protocol

Mixed 1L Agar+LB, Autoclaved on liquid cycle
Let cool while stirring, added 1x Chloroamphenicol
Poured plates, let solidify

Objective: Check and culture from transformations 6/17

Results:

Ligation of Repeat-seq construct to pSB1C3

No colonies on experimental or either control

SLIC of G-block (Csy4 construct) into pSB1C3

No colonies on experimental or either control

SLIC of pDGC3 (pSB1C3-pLac-GFP-mCherry)

~10 colonies on experimental plate
~6 colonies on Insert-only control, none on Backbone-only

SLIC of pSB1C3-J06702 ∆BbsI

~100-200 colonies on experimental plate
~60 colonies on Backbone-only control, none on Insert-only

CCEC Test

Mat of colonies covering plates with 200ng, 20ng, and 2ng plasmid
~2000 colonies covering plate with 0.2ng plasmid
Results: competency 10,000 colonies/ng (high success rate)

Culture colonies overnight from successful SLICs

6 cultures from pDGC3 (pSB1C3-pLac-GFP-mCherry) (labelled 1-6)
6 cultures from pSB1C3-J06702-∆BbsI (labelled 7-12)
All in LB+Cm at 37C

Objective: Build pSB1C3-Repeat-crRNA-Repeat and pSB1C3-rCsy4-sgRNA-rCsy4)

PCR of Repeat-crRNA-Repeat from pdCas9

Oligos 5p-EcoRI-XbaI-Repeat and SpeI-Repeat-3p
Using iGEM stock of Q5 polymerase and buffer
4x50uL reactions, with template 0, 0.4, 0.7, and 1.0 uL
30sec extension, 63C anneal

PCR of G-block (rCsy4-sgRNA-rCsy4)

Oligos pSB1C3-up-1 and pSB1C3-down-1

Using iGEM stock of Q5 polymerase and buffer

4x50uL reactions, with template 0, 0.4, 0.7, and 1.0 uL

Left at -20C for ~1.5 hrs before run because thermocycler in use

30sec extension, 66C anneal

Agarose gel of both PCR products

Lanes 1-4: Repeat-crRNA-Repeat PCR
Lanes 5-8: rCsy4-sgRNA-rCsy4 PCR
Results: strong bands in all six experimental tubes.

Smear upward could indicate problems, but probably correct
Control for Csy4 (Lane 5) alone showed strong band at 1kb, no explanation

gel picture:

PCR cleanup of both PCR products

Qiagen kit
Final concentrations in Elution Buffer (via nanodrop):

Repeat-crRNA-Repeat PCR 30 uL at 131.7 ng/uL
rCsy4-sgRNA-rCsy4 PCR 30 uL at 202 ng/uL

Prep-scale restriction digest of pSB1C3 backbone

Used 70 uL combined from tubes of pSB1C3-J06702-∆BbsI (6/17)
- Shown to be without mutation on 6/17
XbaI/SpeI-HF in cutsmart buffer for 4 hours
extracted larger (2.0kb) band from agarose gel

gel picture:

Gel cleanup of pSB1C3 backbone

Used Qiagen kit instead of Zymoclean (first time)

No isopropanol or sodium acetate necessary
340 mg of gel eluted into 30 uL Elution Buffer

Final concentration 235 ng/uL (via nanodrop)

Gibson Assembly of two plasmids

pSB1C3-rCsy4-sgRNA-rCsy4

100 ng = 0.42 uL pSB1C3 backbone cut with Xbal/SpeI
50 ng = 5xMolar = 0.25 uL G-block PCR

pSB1C3-Repeat-crRNA-Repeat

100 ng = 0.42 uL pSB1C3 backbone cut with Xbal/SpeI
50 ng = 5xMolar = 0.38 uL Repeat-seq PCR

Backbone-only control, insert-only controls for each reaction, positive control
Transformed using iGEM CCEC, Plated on LB+Cm

June 19

Objective: Miniprep and Test 6/18/14 SLIC Colonies

Miniprep Colonies

Qiagen Set
Concentrations of ∆Bbs1 site mutation (7-12) (ng/μL): 192.6, 235.7, 279.3, 239.8, 238.6, 203.8
Concentrations of pDGC3 (1-6) (ng/μL): 229.6, 301.9, 202.3, 206.6, 225.5, 259.1

Test Plasmids with Gel Digest

Gel 1: log-2 ladder
Gel 2-7: ∆Bbs1 site mutation 7-12 tested with EcoR1-HF
Gel 8-13: pDGC3 1-6 tested with Spe1-HF
Gel 14-19: ∆Bbs1 site mutation 7-12 tested with EcoR1-HF and Bbs1
Gel 20-25: pDGC3 1-6 tested with Asc1 and Nhe1-HF

gel picture:

Objective: Observe Ligation Colony Results and Culture Test Colonies

Observe Plates

Csy4-sgRNA-Csy4 Scaffold: 6 colonies

Repeat-seq-Repeat Scaffold: 7 colonies

Gibson positive control: 6 colonies
Backbone-only: 1 colony
Insert only: No colonies

Culture Test Colonies

Cultured 3 Cys4-sgRNA-Cys4 Scaffold colonies
Cultured 3 Repeat-seq-Repeat Scaffold colonies

June 20

Objective: Miniprep and Test 6/19/14 Scaffold gibson clones

Miniprep Colonies

Qiagen Set
Concentrations of Cys4-sgRNA-Cys4 Scaffold (1-3) (ng/μL): 168.4, 165.9, 220
Concentrations of Repeat-seq-Repeat Scaffold (1-3) (ng/μL): 216.9, 84.0, 226.9

Test Plasmids with Gel Digest

Gel 1: log-2 ladder
Gel 2-4: Repeat-seq-Repeat Scaffold 1-3 tested with Acs1 and Nhe1-HF
Gel 5-7: Cys4-sgRNA-Cys4 Scaffold 1-3 tested with EcoR1-HF and Bbs1
Gel 8-10: pDGC3 1-6 tested with EcoR1-HF and Bbs1
Gel 11-13: pDGC3 1-6 tested with EcoR1-HF and Mfe1-HF

gel picture:

Objective: Remove Bbs1 site from mCherry using SLIC

Prepare and Dephosphorylate Bbs1-cut mCherry-pSB1C3

Used official NEB protocol of Calf intestinal protein on Bbs1-cut mCherry-pSB1C3 plasmid
Purified DNA with miniprep with a concentration of 27 ng/μL

SLIC out the Bbs1 site using change oligos

SLIC performed as per standard protocol with the mCherry-∆Bbs1-up and mCherry-∆Bbs1-down oligos on prepared plasmid
Backbone only and insert only controls prepared

Transform into Chemically competent cells

Heat shocked and incubated, then plated as per standard protocol

Objective: Create pDGC3 with Gibson Assembly

Perform Gibson Assembly to create pDGC3 plasmid

Gibson assembly done with pSB1C3 backbone and GFP and mCherry PCR-amplified inserts
Backbone-only, insert-only and positive controls also prepared
Gibson master mix not included on insert-only

Transform assemblies into cells

Heat shocked and incubated, then plated as per standard protocol

June 21

Objective: Remove plates to stop growth

Charlie Cooper

Took plates out of incubator and left at room temperature

June 22

Objective: Choose colonies and incubate cultures

Charlie Cooper

Observe plates

DESCRIPTION?
Choose four colonies to grow over

Incubate Colonies

Colonies grown in LB+Cm overnight

June 23

Objective: Sequence rCsy4-sgRNA-rCsy4 and Repeat-seq-Repeat for BioBrick submission

Prepare plasmid submission using Big Dye protocol

pSB1C3-up and pSB1C3-dn oligoes used
Buchler thermal phaser used to run reaction

Deliver prepped plasmid to DUGSIM

Delivered to lab room in BioSci

Next step: Compare sequence to predicted, submission process

Objective: Test if ∆Bbs1 site was removed

Miniprep Colonies

Qiagen Set
Concentrations of ∆Bbs1 mCherry-pSB1C3 (1-4) (ng/μL): 168.0, 110.2, 175.7, 147.5

Digest mCherry-pSB1C3 plasmid

Standard diagnostic digest protocol used on four overnight colonies from 6/22
Each colony had two digests: EcoR1 and EcoR1/Bbs1

Run and observe gels

Gels run for 17 minutes
Order: 2-log ladder, EcoR1-cut sample 1, EcoR1 and Bbs1-cut sample 1, EcoR1-cut sample 2, EcoR1 and Bbs1-cut sample 2, EcoR1-cut sample 3, EcoR1 and Bbs1-cut sample 3, EcoR1-cut sample 4, EcoR1 and Bbs1-cut sample 4

Objective: Insert sgRNA sequence into rCsy4-sgRNA-rCsy4 Scaffold

Cut Scaffold with Bsa1 enzyme

36 μL of DNA template was cut with 5 μL of enzyme in 10 μL of CutSmart buffer and 49 μL of water
Digested for 2 hours

Qiagen Set clean-up protocol performed
Concentrations of rCsy4-sgRNA-rCsy4 Scaffold (ng/μL): 93.2

Anneal mCherry1 sgRNA from oligos

crRNA-ch1-up and crRNA-ch1-dn oligos annealed using ____ protocol

Ligate into scaffold

Ligate annealed oligos into the cut rCsy4-sgRNA-rCsy4 scaffold using ligation protocol

Insert Tet1 sgRNA into scaffold using SLIC

Used standard SLIC protocol, using scaf-crRNA-Tet1-up and scaf-crRNA-Tet1-dn

Transform into competent cells

Used iGEM stock comp cells
Plated onto LB+Cm plates

Objective: Get pSB1C3 backbone from BBa_R0030

Awaken and Transform R0030 into cells

BBa-R0030 awakened from frozen iGEM supply
Transformed onto iGEM stock competent cells and plated onto LB+Cm

June 24

Objective: Check plates from 6/22/13

Ligation and SLIC of sgRNA

Backbone only and experimental plates have many colonies
Theories are that Bsa1 has degraded or that the plasmid self-ligated
Next step: test Bsa1

Objective: Test Bsa1 effectiveness

Digest test plasmids with Bsa1

TAL 16C, 20A and 20B plasmids used (from last year’s stocks in iGEM Box 3)
Standard analytical restriction digest used with Bsa1 in CutSmart
Left for 2 hours in 37°C

Ran diagnostic gel

1. TAL 16C cut with BsaI (expected 2 fragments around 2.3 kb and one around 5.6 kb)

2. TAL 20A cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)

3. TAL 20B cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)

Results: Bsa1 does cut, but fragments were not exactly expected sizes

16C showed one smeary band ~ 2-3 kb plus a faint, thin band ~ 5-6kb
20A showed two clear bands at ~4-5 kb and ~2-3 kb

Known to have issues, may be incorrect insert

20B showed two clear bands at ~6 kb and 2-3 kb

gel picture:

Objective: Attempt mCherry Bbs1 Mutation

Charlie Cooper tried today.

BsmBI digest:

30uL of 159.1ng/uL pSB1C3-BBa_J06902 (mCherry) digested with BbsI in 100uL reaction & NEBuffer 2.1 for 4h237C. Collected with PCR cleanup protocol and eluted in to 80uL h2o. Ran 2uL on a gel next to undigested DNA at same final concentration. Untreated plasmid runs faster than BbsI-treated, consistent with behavior of un-digested circular DNA.
1: ladder
2: digested plasmid
3: undigested
4,5 unrelated stuff for CBC

CIP:

Added 10uL buffer 3 and 2uL undiluted CIP. 37C for 60m. PCR cleanup again and eluted in to final volume of 35uL with conc ~76 ng/uL

SLIC

1 tube with 1 uL each of SLIC oligos mCherry-Bbs1-left and -rightprepared by speedVac to remove solvent. tubes placed on ice.
T4 reaction set up with 2 uL backbone (0 for insert-only), buffer, bsa and water to 10uL. 0.5 uL T4 poly added and incubated at RT. at 2.5m, tube contents transferred to waiting tube with (or without for bbb-only) dried oligos.
10m on ice, add 60uL comp. DH5alpha, 20m more on ice then 45s heat shock and 2m more on ice before recovering for 60m in SOC and spreading on LB+Cm and 37C overnight

Objective: Build pDGC3 construct

PCR of pLac-GFP-A-Z from pSB1C3-BBa_K741002

4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template

6/16 dilution of 6/14 miniprep

Oligos pSB1C3up and GFP-A-Z-3p
iGEM stock of Q5 polymerase/buffer
iGEM thermocycler protocol with 30 sec extension, 65C anneal

PCR of Asc1-mCherry-Term from pSB1C3-BBa_J06702

4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template

6/16 dilution of 6/14 miniprep

Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3dn
iGEM stock of Q5 polymerase/buffer
iGEM thermocycler protocol with 30 sec extension, 67C anneal

Agarose gel of PCR products:

Successful product in lanes 2 and 4 of GFP, and 2, 3, 4 of mCherry PCR

gel picture:

PCR cleanup of GFP and mCherry amplicons

Accidentally pooled all four tubes of GFP together

Should not be a problem, may reduce yield slightly

Correctly pooled 3 tubes of mCherry
Final concentrations:

Objective: Culture 16 colonies from various plates:

pSB1C3-BBa_B0030

Transformed 6/23

Plate had >100 colonies

Cultured 1 in 5mL LB+Cm

Next steps: use to extract pSB1C3 backbone without gel purification

Gibson products of pDGC3 assembly

Transformed on 6/20
Backbone+Insert plate had ~75 colonies

Cultured 3 in 5mL LB+Cm

Insert-only control plate had ~200-300 colonies

Cultured 2 in 5mL LB+Cm

Backbone-only control had ~15-20 colonies

Cultured 2 in 5mL LB+Cm

Next steps: analytical digests to test if any products were successful, and why insert-only products are so numerous

Insight into how to move forward with troubling construct?

Ligation products of crRNA mCh1 into Repeat-seq scaffold

Transformed on 6/23
Backbone+Insert plate had hundreds of tiny colonies

Cultured 3 in 5mL LB+Cm

Backbone-only control had hundreds of tiny colonies

Cultured 1 in 5mL LB+Cm

Insert-only control had no colonies
Next steps: Test to see if any products were successful, or which false construct caused backbone-only products

SLIC products of crRNA Tet1 into Repeat-seq scaffold

Transformed on 6/23
Backbone+Insert plate had hundreds of tiny colonies

Cultured 3 in 5mL LB+Cm

Backbone-only control had hundreds of tiny colonies

Cultured 1 in 5mL LB+Cm

Insert-only control had no colonies
Next steps: Test to see if any products were successful, or which false construct caused backbone-only products

Objective: Find constitutive GFP constructs for Molecular Titration approach

Transformed four biobricks in pSB1C3 from iGEM kit plates

Plate 1-5I:BBa_K608008

“strong promoter/medium RBS + GFP”

Plate 1-5K:BBa_K608010

“medium promoter/strong RBS + GFP”

Plate 1-5M:BBa_K608011

“medium promoter/medium RBS + GFP”

Plate 1-5O:BBa_K608012

“medium promoter/weak RBS + GFP”

Transformed by heat shock using iGEM CCEC stocks

Plated on LB+Cm

June 25

Objective: Check plates from 6/24/13

Check ∆Bbs1 Plates

Backbone only and experimental plates have many colonies, leading to possibility that CIP did not function

Objective: Check CIP still functions

Digest plasmid with EcoRI

pSB1C3-J06702 ∆Bbs1 plasmids 8 and 9 were used
Standard ligation prep digest preformed on 32 uL of template in CutSmart buffer
Digest cleaned with Qiagen PCR clean-up into 30uL of EB
Concentration of ∆Bbs1 8 was 99.2 ng/uL and 9 was 145.6 ng/uL.

Treat experimental group with Calf Intestinal Phosphotase

15 uL of the cleaned DNA included in standard CIP protocol
CIP cleaned with Qiagen PCR clean-up into 15 uL of EB
Concentration of ∆Bbs1 8 was 32.6 ng/uL and 9 was 62.2 ng/uL.

Ligate plasmids with T4

Ligation protocol used triple amounts of all compounds as standard 100 ng ligations, producing 300 ng of ligated DNA for ∆Bbs1-8, ∆Bbs1-8-CIP, ∆Bbs1-9 and ∆Bbs1-9-CIP in 60 uL
40 uL was cleaned with a Qiagen PCR clean-up into 8.5 uL of EB
20 uL was left to transform into cells

Digest with EcoRV and analyze digestion

8.5 uL of DNA, since concentrations are so low, were used in a restriction digest with 1 uL of CutSmart and 0.5 uL of EcoRV
Ran on gel to test digest
Order: 2-log, ∆Bbs1-8, ∆Bbs1-8-CIP, ∆Bbs1-9, ∆Bbs1-9-CIP

Transform DNA into cells to see rate of uptake

Transformed into iGEM comp cells and plated onto LB+Cm

Objective: Build crRNAs in Repeat-seq scaffold

Miniprep crRNA cultures and BO controls

3 copies crRNA-Tet1 and one BO control

Concentrations in 50 uL (1,2,3,BO):

68, 124.7, 71.4, 89.2 ng/uL

3 copies crRNA-mCh1 and one BO control

Concentrations in 50 uL (1,2,3,BO):

55.8, 51.3, 60.6, 65.1 ng/uL

Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Analytical digest of crRNA-Tet1 minipreps

To determine if minipreps still digestible (if in glycerol, may not be)
4 tubes digested with EcoRI-HF/EcoRV-HF in cutsmart buffer for 2 hrs

Agarose gel of digest results:

1. crRNA-Tet1-1 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

2. crRNA-Tet1-2 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

3. crRNA-Tet1-3 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

4. crRNA-Tet1-BO in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

Results: expected bands present, minipreps still useful in digests

Analytical digest of crRNA minipreps

To test whether new insert is present
8 tubes digested with BsaI/NcoI in cutsmart buffer for 2 hrs
Frozen in iGEM freezer--need to run on gel

Objective: Build pDGC3

MIniprep pDGC3 cultures, BO controls, and IO controls

3 copies pDGC3, 2 copies BO control, 2 copies IO control

Concentrations in 50 uL (1,2,3,BO-1,BO-2,IO-1,IO-2):

55.6, 66.9, 54.1, 32.1, 36.2, 55.5, 58.7 ng/uL

Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Analytical digest of minipreps

To test whether GFP and/or mCherry are present
Two digest each of 7 samples, NdeI/EcoRV-HF and BstEII-HF/EcoRV-HF
All in cutsmart buffer for 2 hrs
Frozen in iGEM freezer--need to run on gel

Objective: Obtain pSB1C3 backbone without gel purification

Miniprep pSB1C3-B0030 culture

Concentration 34 ng/uL (eluted in 50 uL)
Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Prep-scale digest of pSB1C3-B0030 to obtain backbone

Enzymes SpeI-HF/EcoRI-HF in cutsmart buffer
42 uL DNA in 100 uL reaction for 4 hrs
Note: product frozen in iGEM cloning project box overnight
Need to run on gel to confirm and PCR clean to use

Objective: Culture colonies containing constitutive GFP for molecular titration approach

pSB1C3-B0030(as a control with no flourescence)
pSB1C3-K608008“strong promoter/medium RBS + GFP”
pSB1C3-K608010“medium promoter/strong RBS + GFP”
pSB1C3-K608011“medium promoter/medium RBS + GFP”
pSB1C3-K608012“medium promoter/weak RBS + GFP”
To flow tomorrow and save glycerol stocks

June 26

Objective: Check plates from 6/25/13 to confirm CIP activity

Check ∆Bbs1-8 and 9 Plates

Still many colonies on CIP treated plates, but much fewer than non-CIP treated plates
This leads to the conclusion that CIP still functions, but is not completely effective, leading to possible expression of self-ligation in CIP treated ∆Bbs1 experiments

Objective: Dephosphorylate Bbs1 cut pSB1C3-J06702 (mCherry)

Run dephosphorylation

3uL of Cutsmart and 1.5 uL of CIP added to 25 uL of mCherry plasmid, set in 37C for 45 minutes
DNA cleaned with Qiagen PCR Clean-up

Objective: Analyze the constitutive GFP expression of reporters using flow cytometry

Save stocks of Constitutive GFP plasmids

K608008, K608010, K608011, K608012

Flow Cytometry

FSC log 3, 350V
SSC log 3, 350V
B1 log 5, 350V
Order of samples:

1. B0030 (control)

2. K608008

3. K608010

4. K608011

5. K608012

Results: (analyzed 6/30/14)

	E. coli/Simple peak GFP \| Mean (B1-A)	Fold Change from control
pSB1C3-B0030 (control)	0.29	1
pSB1C3-K608008	0.3	1.03
pSB1C3-K608010	201	693.10
pSB1C3-K608011	58.2	200.69
pSB1C3-K608012	118	406.90

Notes:

K608008 showed no fluorescence--could be transformation problem or part problem.
K608010 had messier data for FSC/SSC and for GFP (B-1). Despite having the highest mean, this is not as reliable of a sample.
K608012 had the highest reliable mean, and should be used for at least the initial molecular titration experiments. K608011 is also useful if we need a lower maximum fluorescence level.

June 30

Objective: Insert crRNAs targeting GFP into pdCas9 and Repeat scaffold

Prep-scale Digest of pdCas9

BsaI in Cutsmart buffer
30 uL template in 100 uL reaction
4 hrs at 37C

Anneal crRNA inserts

3 inserts, each annealed with 2 oligos

crRNAs GFP1, GFP2, and GFP3

5uL each oligo in ~boiling water bath, cooled at rt to rt

Ligation of crRNAs into BsaI site in pdCas9 and Repeat scaffold

6 reactions plus 5 controls
pdCas9 with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)

backbone cut with BsaI (6/30), 100ng=3.5uL

pSB1C3-Repeat-scaffold (pDGC1) with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)

backbone cut with BsaI (6/23), 100ng=1uL

BO controls for pdCas9 and Repeat-scaffold
IO controls for all three crRNA inserts
10 uL rxns transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm

Objective: Build pDGC3

Gibson assembly of pSB1C3, GFP, and mCherry

Backbone cut with EcoRI/SpeI on 6/16, 100ng=1.5uL
GFP PCR treated with DpnI on 6/26
mCherry PCR treated with DpnI on 6/26
Two copies, one with 100ng backbone, one with 50ng backbone

1xMolar and 2xMolar inserts (50ng each)

BO and IO controls
Using Mert’s new DIY Gibson mix (15uL into 20uL)
Transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm

Objective: Remove BbsI site from mCherry

SLIC to remove BbsI site from pSB1C3-J06702

Backbone pSB1C3-J06702 super-treated with CIP on 6/26, 100ng=2.5uL
Oligos mCherry-BbsI-left and mCherry-BbsI-right
10 uL rxns transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm

@@ Line 6: / Line 6: @@
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-<td colspan="7"> June 2014 </td>
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-<td colspan="7" align=center><i> Month 3 of Project </i></td>
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-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun1">1</a></td>
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-<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>
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-<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>
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-<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun17">17</a></td>
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-<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>
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 <div class="day">
 <a id="may29"><h2>May 29</h2></a>
@@ Line 339: / Line 190: @@
 <div class="day">
 <a id="jun9"><h2>June 9</h2></a>
-<div class="obj">    </div>
+<div class="obj">Objective: Grow and confirm two backbone plasmids with “RFP coding device” insert from iGEM registry plates</div>
-<div class="ppl">    </div>
+<div class="ppl">Matthew Farnitano and TJ Ciesla</div>
 <div class="lab">
+<p>   Miniprepped and stored glycerol stocks of two copies each of pSB6A1-BBa_J04450 and pSB1C3-BBa_J04450  </p>
+<ul>
+<li>  Glycerol stocks in iGEM bacteria box 1, A3 and A4. </li>
+<li>  Followed Qiagen kit for miniprep procedure </li>
+<li>  All four E. coli strains visibly reddish before miniprep (sign that RFP is expressed) </li>
+<li>  Results: All four successful, concentrations (ng/uL): </li>
+<ul>
+<li>  6A1-1: 429.2, 6A1-2: 461.2,1C3-1: 337.2, 1C3-2: 294.2 </li>
+</ul>
+</ul>
+<p>   </p>
+<ul>
+<li>  10 lanes: </li>
+<ul>
+<li>  1. BBa_J04450+pSB6A1-1(uncut) </li>
+<li>  2. BBa_J04450+pSB1C3-1 (uncut) </li>
+<li>  3. A-1 cut with Kpn1-expected 4.8 and 0.25 kb bands </li>
+<li>  4. A-2 cut with Kpn1-expected 4.8 and 0.25 kb bands </li>
+<li>  5. A-1 cut with Afe1-expected 3.8, 0.9, and 0.28 bands </li>
+<li>  6. A-2 cut with Afe1-expected 3.8, 0.9, and 0.28 bands </li>
+<li>  7. C-1 cut with Kpn1-expected 2.9, 0.25 bands </li>
+<li>  8. C-2 cut with Kpn1-expected 2.9, 0.25 bands </li>
+<li>  9. C-1 cut with Nco1-expected 1.7, 1.4 bands </li>
+<li>  10. C-2 cut with Nco1-expected 1.7, 1.4 bands </li>
+</ul>
+</ul>
+<p>   Results: All expected bands are present in all samples. A faint unexpected band is present at ~2.5-3.0 kb in lanes 3, 4, 5, 6, maybe 7 and 8. Band is not strong enough to be a full piece of the plasmid </p>
+<p>   Gel picture:  </p>
+<a href="https://static.igem.org/mediawiki/2014/7/76/Image13.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/76/Image13.png"></a>
+<!--https://2014.igem.org/File:Image13.png or https://static.igem.org/mediawiki/2014/7/76/Image13.png -->
+<p>   Note: Kept copies 6A1-2 (glycerol box A-3) and 1C3-1 (glycerol box A-4), discarded others.
+TJ Ciesla and Charlie Cooper streaked a plate from 1C3-1 for use later </p>
+<div class="obj">Objective: Grow and confirm two backbone plasmids with “RFP coding device” insert from iGEM registry plates</div>
+<div class="ppl">Matthew Farnitano and TJ Ciesla</div>
+<p>   Ligation-scale restriction digest of pSB1C3-BBa_J04450 and 6/6/14 PCR product </p>
+<ul>
+<li>  EcoRI-HF and SpeI-HF (in Cutsmart buffer) </li>
+<li>  35 uL pSB1C3 DNA (337.2 ng/uL) in 50 uL reaction for 3 hrs </li>
+<li>  35 uL Repeat-gRNA-Repeat PCR product in 50 uL reaction for 3 hrs </li>
+</ul>
+<p>   Made 0.8% agarose TAE gel to separate pSB1C3 backbone from insert </p>
+<p>   Ran pSB1C3-BBa_J04450 digest on gel </p>
+<ul>
+<li>  Expected to see two bands, 2.0 kb and 1.1 kb.  </li>
+</ul>
+<p>   Results: only one large band ~3.0 kb with faint bands slightly below</p>
+<p> Gel photo: </p>
+<a href="https://static.igem.org/mediawiki/2014/1/18/Image11.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/1/18/Image11.png"></a>
+<!--https://2014.igem.org/File:Image11.png or https://static.igem.org/mediawiki/2014/1/18/Image11.png -->
 </div>
 </div>
@@ Line 349: / Line 249: @@
 <div class="day">
 <a id="jun10"><h2>June 10</h2></a>
-<div class="obj">    </div>
+<div class="obj">Objective: Assemble Repeat-gRNA-Repeat scaffold in pSB1C3 backbone</div>
-<div class="ppl">    </div>
+<div class="ppl">Matthew Farnitano and TJ Ciesla</div>
 <div class="lab">
+<p>   Analytical Restriction Digest </p>
+<ul>
+<li>  To test why digest 6/9/14 did not work as expected </li>
+<li>  pSB1C3-BBa_J04450 in three cuts, with pCC415 as a control cut (see gel results below) </li>
+</ul>
+<p>   Agarose gel of restriction digest</p>
+<ul>
+<li>  1. pCC415 cut with SacI-HF: expecting two bands </li>
+<li>  2. pSB1C3-BBa_J04450 cut with SacI-HF and SpeI-HF: expecting 2.0 and 1.1 kb bands </li>
+<li>  3. pSB1C3-BBa_J04450 cut with SacI-HF and EcoRI-HF: expecting 2.3 and 0.9 kb bands </li>
+<li>  4. pSB1C3-BBa_J04450 cut with SpeI-HF and EcoRI-HF: expecting 2.0 and 1.1 kb bands </li>
+</ul>
+<p>   Results: Expectations met</p>
+<p>   Gel photo:</p>
+<!--https://2014.igem.org/File:Image25.png or https://static.igem.org/mediawiki/2014/c/c3/Image25.png -->
+<a href="https://static.igem.org/mediawiki/2014/c/c3/Image25.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c3/Image25.png"></a>
+<p>   Isolate DNA from restriction digest of PCR product (Repeat-seq-Repeat)</p>
+<ul>
+<li>  DNA isolated using Qiagen Spin columns - last few steps of miniprep protocol </li>
+<li>  DNA is 75 ng/µL in 30 µL EB buffer </li>
+</ul>
+<div class="obj">Objective: Grow plasmids</div>
+<p>   Grow bacteria in medium</p>
+<ul>
+<li>  1. DH5α (Charlie’s) in SOC for future transformation </li>
+<li>  2. DH5α, plate 3, 15B in LB+CM* (pSB1C3-BBa_J06702) </li>
+<li>  3. DH5α, plate 2, 6F in LB+CM* (pSB1C3-BBa_R0040)</li>
+<li>  4. DH5α, plate 2, 17M in LB + CM* (pSB1C3-BBa_K741002) </li>
+<li>  5. DH5α, pSB1C3-BBa-J04450 in LB + CM**  </li>
+<li>  6. DH5α, pSB6A1-BBa-J04450 in LB + amp**  </li>
+<li>  *2, 3, 4 contain plasmids w/biobricks </li>
+<li>  **5 and 6 will be used to calibrate flow cytometry in 6/11/14 </li>
+</ul>
+<p>   Next steps:</p>
+<ul>
+<li>  Miniprep, digest to confirm plasmids from 2, 3, 4 </li>
+<ul>
+<li>  J06702 contains RBS-mCherry-T, to be PCRed out </li>
+<li>  R0040 contains pTet, to be PCRed out </li>
+<li>  K741002 contains pLac-RBS-GFP, and Terminator, both to be PCRed out  </li>
+</ul>
+<li>  Dilute 5 and 6, use for flow calibration </li>
+</ul>
 </div>
 </div>
@@ Line 359: / Line 302: @@
 <div class="day">
 <a id="jun11"><h2>June 11</h2></a>
-<div class="obj">    </div>
+<div class="obj">Objective: Prepare chemically competent E. coli (DH5α)</div>
-<div class="ppl">    </div>
 <div class="lab">
+<p>   Froze 6 1mL seed stocks of DH5alphas in 15% glycerol</p>
+<ul>
+<li>  From 6/10 culture </li>
+<li>  For use in iGEM Chemically Competent Cell protocol </li>
+</ul>
+<p>   Grow 2 seed stocks in SOC overnight for CCEC protocol</p>
+<ul>
+<li>  Each stock 1mL DH5alpha in 250mL SOC </li>
+<li>  In shaker at room temperature (started 6:30pm) </li>
+</ul>
+<p>   Next Steps:</p>
+<ul>
+<li>  Complete protocol with cultures </li>
+</ul>
+<div class="obj">Objective: Prepare biobrick plasmids for cloning</div>
+<p>   Froze 15% glycerol stocks of three biobrick plasmids at -80C</p>
+<ul>
+<li>  DH5alpha, pSB1C3-BBa_K741002, stock location Box 1-A5 </li>
+<li>  DH5alpha, pSB1C3-BBa_J06702, stock location Box 1-A6 </li>
+<li>  DH5alpha, pSB1C3-BBa_R0040, stock location Box 1-A7 </li>
+</ul>
+<p>   Miniprepped three plasmids</p>
+<ul>
+<li>  Standard Qiagen kit protocol </li>
+<li>  Results:  </li>
+<ul>
+<li>  1C3-K741002 40uL at 98 ng/uL, iGEM box 1 A5 </li>
+<li>  1C3-J06702 40uL at 114 ng/uL, iGEM box 1 A6 </li>
+<li>  1C3-R0040 40uL at 170 ng/uL, iGEM box 1 A7 </li>
+</ul>
+</ul>
+<p>   Analytical restriction digest of minipreps to confirm</p>
+<ul>
+<li>  Each of three plasmids in EcoRI-HF/SpeI-HF and in PstII-HF  </li>
+<ul>
+<li>  All in cutsmart buffer, 2 hrs at 37C </li>
+</ul>
+</ul>
+<p>   Agarose gel of restriction digest</p>
+<ul>
+<li>  1. 1C3-K741002 in EcoRI-HF/SpeI-HF : expected 2.0, 1.1 kb bands </li>
+<li>  2. 1C3-K741002 in PvuII-HF : expected 1.9, 0.89, 0.4 kb bands </li>
+<li>  3. 1C3-J06702 in EcoRI-HF/SpeI-HF : expected 2.0, 0.89 kb bands </li>
+<li>  4. 1C3-J06702 in PvuII-HF : expected 2.0, 1.0 kb bands </li>
+<li>  5. 1C3-R0040 in EcoRI-HF/SpeI-HF : expected 2.0, 0.08 kb bands </li>
+<li>  6. 1C3-R0040 in PvuII-HF : expected 2.1 kb band </li>
+</ul>
+<p>   Results: Order of tubes in gel was not recorded. Lanes probably good, but should retest to be sure.</p>
+<p>  Gel photo:</p>
+<!-- https://2014.igem.org/File:Image20.png or https://static.igem.org/mediawiki/2014/a/a1/Image20.png -->
+<a href="https://static.igem.org/mediawiki/2014/a/a1/Image20.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/a1/Image20.png"></a>
+<p>   Next steps:</p>
+<ul>
+<li>  Reconfirm plasmids </li>
+<li>  PCR pLac-RBS-GFP from K741002 </li>
+<li>  PCR RBS-mCherry-Term from J06702 </li>
+<li>  Isolate pSB1C3 </li>
+<li>  SLIC to make pDGC3 </li>
+</ul>
+<div class="obj">Objective: Prepare biobrick plasmids for cloning</div>
+<p>   Diluted E. coli to grow to log phase for flow</p>
+<ul>
+<li>  DH5alpha (SOC), pSB1C3-BBa_J04450 (LB+Cm), and pSB6A1-BBaJ04450 (LB+Amp) </li>
+<li>  Each strain three dilutions: 1/100, 1/250, 1/500 </li>
+<li>  Growing from ~10am </li>
+<li>  Note: Overnight culture of 1C3 had no visible red, but culture of 6A1 was strong reddish-pink. Possible that RFP is not expressed well in 1C3. </li>
+</ul>
+<p>   Flow Cytometry Parameter setting</p>
+<ul>
+<li>  1. calibration beads </li>
+<li>  2. messing around with parameters  </li>
+<li>  3. pSB6A1-BBa_J04450 1/500 initial dilution </li>
+<li>  4. pSB1C3-BBa_J04450 1/500 initial dilution, 1/50 final dilution at flow time </li>
+<li>  5. DH5alpha 1/500 initial dilution, 1/50 final dilution at flow time </li>
+<li>  6. pSB6A1-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time </li>
+<li>  7. pSB1C3-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time </li>
+</ul>
+<p>   Notes: pSB1C3-BBa_J04450 showed strong, clear red fluorescence at both dilutions. pSB6A1 showed a bimodal fluorescence with some cells matching 1C3 fluorescence and others matching DH5alpha natural fluorescence. All samples showed two peaks of FS:SS data. These peaks did not correlate with the bimodal fluorescence seen in 6A1. </p>
+<p>   Parameters settled on (subject to change):
+FS: 350 volts, log 3
+SS: 350 volts, log 3
+Y2: 400 volts, log 5</p>
+<p>   Next Steps: </p>
+<ul>
+<li>  Perform formal data analysis  </li>
+</ul>
+<div class="obj">Objective: Create Csy4-gRNA-Csy4 Scaffold using G-block</div>
+<p>   G-block arrived today, CC split with Sargis</p>
+<p>   PCR of G-block to obtain iGEM piece</p>
+<ul>
+<li>  Used oligos pSB1C3-up and pSB1C3-down </li>
+<li>  iGEM protocol with 65C anneal, 20 sec extension </li>
+</ul>
+<p>   Agarose Gel of PCR product</p>
+<ul>
+<li>  Used HindIII ladder instead of 2-log </li>
+<li>  Results looked good, hard to tell exactly with ladder </li>
+<li>  Some junk in no template control, but absent in other tubes </li>
+</ul>
+<p>   Gel photo: </p>
+<!--https://2014.igem.org/File:GEL_6-11-14_MCF.png or https://static.igem.org/mediawiki/2014/7/74/GEL_6-11-14_MCF.png-->
+<a href="https://static.igem.org/mediawiki/2014/7/74/GEL_6-11-14_MCF.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/74/GEL_6-11-14_MCF.png"></a>
+<p>   PCR cleanup of G-block PCR</p>
+<ul>
+<li>  Results: 30 uL product at 167.6 ng/uL  </li>
+</ul>
+<p>   SLIC of pSB1C3 and Csy4-gRNA-Csy4 and Transformed</p>
+<ul>
+<li>  1 uL pSB1C3 from gel, purified (today), cut with EcoRI/SpeI </li>
+<li>  0.2 uL g-block in 10 uL reaction </li>
+<li>  One no-backbone control (not enough backbone for no-insert control </li>
+<li>  Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm </li>
+</ul>
+<div class="obj">Objective: Create Repeat-seq-Repeat Scaffold </div>
+<p>   Gel clean-up of pSB1C3 cut from EcoRI/SpeI gel</p>
+<p>   Gel picture:</p>
+<!--https://2014.igem.org/File:6-11-14-3.png or https://static.igem.org/mediawiki/2014/7/70/6-11-14-3.png-->
+<a href="https://static.igem.org/mediawiki/2014/7/70/6-11-14-3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/70/6-11-14-3.png"></a>
+<ul>
+<li>  Digested and cut 6/10 and frozen overnight </li>
+<li>  Used Zymoclean prep kit </li>
+<li>  Results: Obtained 10 uL product at 133.1 ng/uL </li>
+</ul>
+<p>   Ligation of pSB1C3 with Repeat-seq-Repeat and Transform</p>
+<ul>
+<li>  4 uL pSB1C3 backbone from gel purified today </li>
+<li>  2 uL Repeat-seq-Repeat insert cut 6/9, cleaned 6/10 </li>
+<li>  One no-insert control (next time do a no-backbone control as well) </li>
+<li>  Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm </li>
+</ul>
 </div>
 </div>
@@ Line 369: / Line 440: @@
 <div class="day">
 <a id="jun12"><h2>June 12</h2></a>
-<div class="obj">    </div>
+<div class="obj">Objective: Create Repeat-seq-Repeat construct and Csy4-gRNA-Csy4 construct</div>
-<div class="ppl">    </div>
 <div class="lab">
+<p>   Results of transformation: no colonies on SLIC or Ligation plate </p>
+<ul>
+<li>  Controls also blank </li>
+<li>  Made new backbone for use (see below) </li>
+</ul>
+<p>   Overnight ligation of Repeat-seq-Repeat PCR with pSB1C3/p>
+<ul>
+<li>  ~100 ng backbone (2 uL) with ~30 ng insert (0.5 uL) </li>
+<ul>
+<li>  in 20 uL reaction </li>
+</ul>
+<li>  Two controls: one with no backbone, one with no insert </li>
+<li>  Left in heat block in cold room (16C) at 6pm for overnight ligation </li>
+</ul>
+<p>   Next steps: </p>
+<ul>
+<li>  Do a parallel room temp. ligation </li>
+<li>  SLIC of Csy4 construct </li>
+<li>  Transform all of the above, use Charlie’s CCEC until ours are confirmed </li>
+</ul>
+<div class="obj">Objective: Obtain pSB1C3 backbone (various cloning uses)</div>
+<p>   Restriction digest of pSB1C3-K741002 and pSB1C3-J06702</p>
+<ul>
+<li>  25 uL each backbone in 100 uL reactions </li>
+<li>  SpeI-HF and EcoRI-HF in Cutsmart buffer (11am start) </li>
+</ul>
+<p>   Agarose gel of product for extraction</p>
+<ul>
+<li>  One lane looked as expected (2.0 and 0.8 kb bands) </li>
+<li>  Other lane had 2.0 kb backbone band but no insert band </li>
+<ul>
+<li>  possibly a faint band ~.1 kb </li>
+</ul>
+<li>  Extracted backbone (2.0 kb band) from both lanes separately </li>
+</ul>
+<p>  Gel photo: </p>
+<!--https://2014.igem.org/File:6-12-14-1.png or https://static.igem.org/mediawiki/2014/a/ae/6-12-14-1.png -->
+<a href="https://static.igem.org/mediawiki/2014/a/ae/6-12-14-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/ae/6-12-14-1.png"></a>
+<p>   Gel purification of pSB1C3 </p>
+<ul>
+<li>  Used Zymoclean prep kit </li>
+<li>  Final elution:  </li>
+<ul>
+<li>  Confirmed piece 20 uL at ng/uL (teal tube) </li>
+<li>  Unconfirmed piece 20 uL at ng/uL (orange tube) </li>
+</ul>
+<li>  Notes: it is possible that the lack of insert is due to mislabelled tubes. Analytical digests and reculturing of the three plasmids in pSB1C3 (K741002, J06702, R0040) will determine if this is the case and correct any mislabelling.  </li>
+</ul>
+<div class="obj">Objective: Create pDGC3</div>
+<p>   PCR of pLac-RBS-GFP from pSB1C3-K741002</p>
+<ul>
+<li>  With oligos pSB1C3-up and GFP-A-Z-3p </li>
+<li>  Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL) </li>
+<li>  iGEM protocol with 30 sec extension, 65C anneal </li>
+</ul>
+<p>   PCR of RBS-mCherry-Term from pSB1C3-J06702</p>
+<ul>
+<li>  With oligos 5p-Z-Asc1-RBS-mCherry </li>
+<li>  Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL) </li>
+<li>  iGEM protocol with 30 sec extension, 67C anneal </li>
+</ul>
+<p>   Agarose gel of PCR results</p>
+<ul>
+<li>  Lanes 1-4: pSB1C3-K741002 PCR </li>
+<ul>
+<li>  No product (fuzzy primer bands only) </li>
+</ul>
+<li>  Lanes 5-8: pSB1C3-J06702 PCR </li>
+<ul>
+<li>  Some product present at low concentrations </li>
+</ul>
+</ul>
+<p>   Gel photo:</p>
+<!--https://2014.igem.org/File:6-12-14-2.png or https://static.igem.org/mediawiki/2014/0/06/6-12-14-2.png -->
+<a href="https://static.igem.org/mediawiki/2014/0/06/6-12-14-2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/06/6-12-14-2.png"></a>
+<p>   PCR cleanup of pSB1C3-J06702 PCR</p>
+<ul>
+<li>  To see if low concentrations are enough to work with </li>
+<ul>
+<li>  <50 ng/uL present, need to redo PCR </li>
+</ul>
+<li>  Next steps: Redo PCR (once proper plasmids are confirmed), confirm, cleanup, SLIC  </li>
+</ul>
+<div class="obj">Objective: Prepare Chemically Competent E. coli</div>
+<p>   At ~11am, OD600 absorption rate for the cultures was .127 and .137</p>
+<ul>
+<li>  Aiming for OD600 of 2-3 </li>
+<li>  Let grow for 50 more minutes (approx. one doubling time) </li>
+<li>  OD600 still low (.163, .178), let grow another hour </li>
+<li>  OD600 >0.2 </li>
+</ul>
+<p>   Completed protocol as stated from iGEM website</p>
+<ul>
+<li>  Used conical-bottom tubes instead of flat-bottom, resulting in difficulty of resuspending cell pellets. Had to vortex pellets substantially to resuspend, which may reduce competence. </li>
+<li>  50-ml tubes used were smaller than those used by iGEM headquarters. Result was more tubes, each with suggested volume, so final culture was too dilute (OD < .1). Pelleted and resuspended in half the volume </li>
+<li>  Final volume 50 uL (for a double batch), in 50 tubes  </li>
+<li>  Tubes in -80C new box, “iGEM chemically competent cells” </li>
+</ul>
+<p>   Next steps: Test competence with transformation of known circular plasmid</p>
+<div class="obj">Objective: Reconfirm plasmids to check for mislabelling</div>
+<p>   Streaked single plate with frozen stocks of three plasmids in pSB1C3</p>
+<ul>
+<li>  K741002, J06702, R0040 </li>
+<li>  All on same LB+Cm plate, because it was the last one </li>
+<ul>
+<li>  Need to make more </li>
+</ul>
+<li>  Incubated overnight at 37C </li>
+</ul>
+<p>   Next steps: </p>
+<ul>
+<li>  Culture, miniprep and digest to confirm correct insert </li>
+<li>  Digest current miniprep DNA as well </li>
+<li>  Re-label tubes if necessary </li>
+</ul>
 </div>
 </div>
@@ Line 379: / Line 563: @@
 <div class="day">
 <a id="jun13"><h2>June 13</h2></a>
-<div class="obj">    </div>
 <div class="lab">
+<p class="obj">Objective: Reconfirm plasmids in pSB1C3 backbone (possible mislabelling event)</p>
+Analytical Restriction Digest of 3 plasmids (2 digests each)
+<ul>
+<li>Each plasmid in PvuII-HF and in EcoRI-HF/SpeI-HF</li>
+<li>All reactions in cutsmart buffer</li>
+<li>Note that plasmid names are as labelled, might not be what is present</li>
+</ul>
+<p>Agarose gel of results:</p>
+<p>1. pSB1C3-BBa_K741002 tube in PvuII-HF </p>
+<p>2. pSB1C3-BBa_J06702 tube in PvuII-HF </p>
+<p>3. pSB1C3-BBa_R0040 tube in PvuII-HF </p>
+<p>4. pSB1C3-BBa_K741002 tube in EcoRI-HF/SpeI-HF </p>
+<p>5. pSB1C3-BBa_J06702 tube in EcoRI-HF/SpeI-HF</p>
+<p>6. pSB1C3-BBa_R0040 tube in EcoRI-HF/SpeI-HF</p>
+<p>Results: No DNA in all K74 and J06 tubes. Tubes labelled R0040 appear to match expected for J06702</p>
+<ul>
+<li>K74 and J06 were 1 ng/uL dilutions, so DNA was likely too dilute to show up</li>
+</ul>
+<ul>
+<li>Hypothesis: R0040 and J06702 tubes are flipped. Possible that K741002 tube is incorrect as well</li>
+<li>gel picture:</li>
+</ul>
+<!--https://2014.igem.org/File:6-13-14-1.jpg or https://static.igem.org/mediawiki/2014/0/02/6-13-14-1.jpg-->
+<a href="https://static.igem.org/mediawiki/2014/0/02/6-13-14-1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/02/6-13-14-1.jpg"></a>
+<p class="obj">Objective: Create pDGC3 using biobrick parts</p>
+<p>
+PCR of pLac-RBS-GFP from BBa_K741002</p>
+<ul>
+<li>8 tubes, with two templates in 4 tubes each (0, .4, .7, 1.0 uL template)</li>
+</ul>
+<ul>
+<li>&ldquo;K&rdquo; template is tube labelled as BBa_K741002 (1 ng/uL dilution)</li>
+<li>&ldquo;J&rdquo; tube is tube labelled as BBa_J06702 (1 ng/uL dilution)</li>
+</ul>
+<ul>
+<li>Trying in case this tube is mislabelled and contains K74</li>
+</ul>
+<ul>
+<li>Oligos pSB1C3up and GFP-A-Z-3p</li>
+<li>iGEM protocol with 30 sec extension time and 65C anneal</li>
+</ul>
+<p>PCR of RBS-mCherry-Term from BBa_J06702</p>
+<ul>
+<li>4 tubes at 0, .4, .7, and 1.0 uL template</li>
+</ul>
+<ul>
+<li>Used tube labelled &ldquo;R&rdquo; as template</li>
+</ul>
+<ul>
+<li>Suspect that this tube is mislabelled and contains J06</li>
+</ul>
+<ul>
+<li>Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3-down</li>
+<li>iGEM protocol with 30 sec extension time and 67C anneal</li>
+</ul>
+<p>Agarose gel of PCRs</p>
+<p>Lanes 1-4: K741002 PCR with &ldquo;K&rdquo; template</p>
+<p>Lanes 5-8: K741002 PCR with &ldquo;J&rdquo; template</p>
+<p>Lanes 9-12: J06702 PCR with &ldquo;R&rdquo; template</p>
+<ul>
+<li>Results: primer bands only on all K741002 lanes. Strong expected band in lanes 10-12 indicating succesful PCR of J06702 </li>
+</ul>
+<ul>
+<li>Further evidence that &ldquo;R&rdquo; is mislabelled and contains &ldquo;J&rdquo;</li>
+</ul>
+<p>gel picture:</p>
+<!--https://2014.igem.org/File:GEL_6-13-14_2.png or https://static.igem.org/mediawiki/2014/7/78/GEL_6-13-14_2.png-->
+<a href="https://static.igem.org/mediawiki/2014/7/78/GEL_6-13-14_2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/78/GEL_6-13-14_2.png"></a>
+<p>PCR cleanup of J06702 PCR</p>
+<p class="obj">Objective: Remove BbsI site from mCherry in J06702</p>
+<p>Restriction digest of tube labelled R0040</p>
+<ul>
+<li>Hopefully tube contains J06702 as predicted earlier</li>
+<li>With Bbs1 in buffer 2.1 with BSA</li>
+</ul>
+<ul>
+<li>50 uL reaction with 25 uL DNA</li>
+</ul>
+<p>Agarose gel of restriction digest</p>
+<ul>
+<li>Results: Looks as expected, hard to tell with single band</li>
+<li> gel photo: </li>
+</ul>
+<!--https://2014.igem.org/File:GEL_6-13-14_MCF_bbs1.png or https://static.igem.org/mediawiki/2014/2/25/GEL_6-13-14_MCF_bbs1.png" -->
+<a href="https://static.igem.org/mediawiki/2014/2/25/GEL_6-13-14_MCF_bbs1.png" target="_blank"> <img src="https://static.igem.org/mediawiki/2014/2/25/GEL_6-13-14_MCF_bbs1.png"></a>
+<p>PCR cleanup of Restriction digest</p>
+<ul>
+<li>Eluted in 30 uL </li>
+<li>Still need to check concentration</li>
+<li>Frozen at -20C</li>
+</ul>
+<p class="obj">Objective: Create Repeat-seq-Repeat construct in pSB1C3</p>
+<p>Ligation of Repeat-seq-Repeat PCR to pSB1C3 (confirmed green tube)</p>
+<ul>
+<li>Three identical tubes: &ldquo;controls&rdquo; actually have all pieces in them</li>
+<li>20 uL reactions with 2 uL (100 ng) backbone and 0.5 uL (30 ng) insert</li>
+<li>Room temp for 1 hour</li>
+</ul>
+<p>Transformation of ligations from overnight (6/12) and rt (6/13)</p>
+<ul>
+<li>6 total tubes: 4 experimental and 2 controls (both from overnight)</li>
+</ul>
+<p class="obj">Objective: Reconfirm and miniprep from frozen stocks of 3 plasmids in pSB1C3</p>
+<p>Cultured 2 colonies each from 3 plasmid strains on 6/12/14 plate</p>
+<ul>
+<li>pSB1C3-K741002, pSB1C3-J06702, and pSB1C3-R0040</li>
+</ul>
+<p>TODO for weekend: </p>
+<ul>
+<li>Miniprep cultures from frozen stock reconfirmations (Sat)</li>
+<li>Pick and culture colonies from 6 ligation plates (Sat)</li>
+<li>Miniprep cultures from ligation plates (Sun)</li>
+</ul>
 </div>
 </div>
@@ Line 392: / Line 735: @@
 <div class="lab">
+<p>Gblock SLIC in to pSB1C3 conducted by CBC</p>
+<ul>
+<li><p class="obj">Objective: gRNA scaffold for 1) submission as a biobrick and 2) use for generating Csy4/ gRNA arrays</p></li></ul>
+<ul>
+<li></li></ul><a href="#" name="0a12efe8d44ed0181bce9f64d3a7def7c41d6b31"></a><a href="#" name="0"></a><table cellpadding="10" cellspacing="0"><tbody><tr><td><p></p></td><td><p>backbone + insert</p></td><td><p>bb only</p></td><td><p>io</p></td></tr><tr><td><p>digested &amp; purified backbone at ~50ng/uL</p></td><td><p>2</p></td><td><p>2</p></td><td><p>0</p></td></tr><tr><td><p>PCRd gBlock </p></td><td><p>1</p></td><td><p>0</p></td><td><p>1</p></td></tr><tr><td><p>buffer 2</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>10x BSA</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>water</p></td><td><p>5</p></td><td><p>6</p></td><td><p>7</p></td></tr></tbody></table><ul>
+<li>all but enzyme pre-mixed in 500uL eppies. enzyme added and incubate 2.5m at RT before icing tubes for 10m. 65 uL CBC&rsquo;s chem. comp DH5alpha then 20m on ice, 45s 42C shock, 2m on ice, 60m recovery on SOC then spread on LB+Cm overnight.</li>
+<li>Results:&nbsp;no colonies the next day on any plates. </li></ul><p></p><p>SLIC repair of BbsI cut site in mCherry by CBC</p><ul>
+<li>
+<p class="obj">Objective: BbsI/golden gate compatible mCherry for gRNA or crRNA array assembly schemes</p></li></ul>
+<ul>
+<li>1 uL each of mCherry-Bbs1-left&nbsp;and mCherry-Bbs1-right&nbsp;at 100uM added to 500uL eppies and evaporated in SpeedVac for backbone + insert and insert-only control. placed on ice to pre-cool</li>
+<li></li></ul><a href="#" name="33631ef86d6ca3ad7971f6da6fd9ca95e72eeacd"></a><a href="#" name="1"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p></p></td><td><p>backbone + insert</p></td><td><p>bb only</p></td><td><p>io</p></td></tr><tr><td><p>bbsi-dig backbone (~5 0ng/uL)</p></td><td><p>2</p></td><td><p>2</p></td><td><p>0</p></td></tr><tr><td><p>oligos (evapd)</p></td><td><p>2</p></td><td><p>0</p></td><td><p>2</p></td></tr><tr><td><p>buffer 2</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>10x BSA</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>water</p></td><td><p>6</p></td><td><p>6</p></td><td><p>8</p></td></tr></tbody></table><ul>
+<li>backbone + insert and bb only reactions treated with T4 poly as one 2x volume, then at 2.5m at RT 10 uL transferred to pre-chilled backbone + insert tube with waiting oligos. insert only reaction run as DNA-free dummy reaction for 2.5m in enzyme then transferred to pre-chilled tube with oligos. </li>
+<li>65 uL chemically competent DH5alpha (from cbc) added at 10m on ice, incubated 20m, then heat shocked 45m at 42C then 2m on ice again before recovering for 60m in SOC and spread on plates.</li>
+<li>Results: &gt; 100 colonies on backbone-only control. improper spreading of backbone + insert makes it hard to tell, but there may be slightly more colonies there than on the bb-only.</li>
+</ul>
 </div>
 </div>
@@ Line 422: / Line 786: @@
 <li> Gel picture:</li>
 </ul>
+<!--https://2014.igem.org/File:6-16-14-1.png or https://static.igem.org/mediawiki/2014/5/50/6-16-14-1.png -->
+<a href="https://static.igem.org/mediawiki/2014/5/50/6-16-14-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/50/6-16-14-1.png"></a>
 <p>Objective: Obtain pSB1C3 backbone for various transformations</p>
 <p>Prep-scale digest of pSB1C3-BBa_K741002</p>
@@ Line 449: / Line 816: @@
 <li>Gel picture:</li>
 </ul>
+<!--https://2014.igem.org/File:6-16-14-2.png or https://static.igem.org/mediawiki/2014/9/9c/6-16-14-2.png -->
+<a href="https://static.igem.org/mediawiki/2014/9/9c/6-16-14-2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9c/6-16-14-2.png"></a>
 <p>Objective: Remove BbsI site from mCherry</p>
 <p> SLIC transformations from 6/15(Charlie) showed colonies on the experimental plate as well as on the no-insert control</p>
@@ Line 462: / Line 834: @@
 <li>Gel picture:</li>
 </ul>
+<!--https://2014.igem.org/File:6-16-14-3.png or https://static.igem.org/mediawiki/2014/f/ff/6-16-14-3.png-->
+<a href="https://static.igem.org/mediawiki/2014/f/ff/6-16-14-3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/ff/6-16-14-3.png"></a>
 <p>PCR cleanup of BbsI cut BBa_J06702</p>
 <ul>
@@ Line 481: / Line 858: @@
 <li> Gel picture:</li>
 </ul>
+<!--https://2014.igem.org/File:6-16-14-4.png or https://static.igem.org/mediawiki/2014/f/f9/6-16-14-4.png-->
+<a href="https://static.igem.org/mediawiki/2014/f/f9/6-16-14-4.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/f9/6-16-14-4.png"></a>
 <p>PCR cleanup</p>
 <ul>
@@ Line 542: / Line 924: @@
 <li>gel picture:</li>
 </ul>
+<!-- https://2014.igem.org/File:GEL_MCF_6-17-14.png or https://static.igem.org/mediawiki/2014/5/57/GEL_MCF_6-17-14.png-->
+<a href="https://static.igem.org/mediawiki/2014/5/57/GEL_MCF_6-17-14.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/57/GEL_MCF_6-17-14.png"></a>
 <p>Objective: Create various plasmids by Ligation/SLIC</p>
 <p> Ligation to create pSB1C3-Repeat-seq scaffold</p>
@@ Line 661: / Line 1,046: @@
 </ul>
 <p>gel picture:</p>
+<!--https://2014.igem.org/File:6-18-14-1.png or https://static.igem.org/mediawiki/2014/e/e6/6-18-14-1.png-->
+<a href="https://static.igem.org/mediawiki/2014/e/e6/6-18-14-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e6/6-18-14-1.png"></a>
 <p>PCR cleanup of both PCR products</p>
@@ Line 681: / Line 1,068: @@
 </ul>
 <p>gel picture:</p>
+<!--https://2014.igem.org/File:6-18-14-2.png or https://static.igem.org/mediawiki/2014/f/fa/6-18-14-2.png-->
+<a href="https://static.igem.org/mediawiki/2014/f/fa/6-18-14-2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/fa/6-18-14-2.png"></a>
 <p>Gel cleanup of pSB1C3 backbone</p>
@@ Line 729: / Line 1,119: @@
 </ul>
 <p>    gel picture:</p>
+<!--https://2014.igem.org/File:GEL_Mcf_6-19-14.png or https://static.igem.org/mediawiki/2014/6/6f/GEL_Mcf_6-19-14.png-->
+<a href="https://static.igem.org/mediawiki/2014/6/6f/GEL_Mcf_6-19-14.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/6/6f/GEL_Mcf_6-19-14.png"></a>
 <p>Objective: Observe Ligation Colony Results and Culture Test Colonies</p>
@@ Line 769: / Line 1,162: @@
 </ul>
 <p>gel picture:</p>
+<!--https://2014.igem.org/File:GEL_6-20-14_MCF.png or https://static.igem.org/mediawiki/2014/a/a6/GEL_6-20-14_MCF.png-->
+<a href="https://static.igem.org/mediawiki/2014/a/a6/GEL_6-20-14_MCF.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/a6/GEL_6-20-14_MCF.png"></a>
 <p>Objective: Remove Bbs1 site from mCherry using SLIC</p>
@@ Line 828: / Line 1,224: @@
 </div>
+<div class="day">
+<a id="jun23"><h2>June 23</h2></a>
+<div class="lab">
+<p class="obj">Objective: Sequence rCsy4-sgRNA-rCsy4 and Repeat-seq-Repeat for BioBrick submission</p>
+<p>Prepare plasmid submission using Big Dye protocol</p>
+<ul>
+<li>pSB1C3-up and pSB1C3-dn oligoes used</li>
+<li>Buchler thermal phaser used to run reaction</li>
+</ul>
+<p>Deliver prepped plasmid to DUGSIM</p>
+<ul>
+<li>Delivered to lab room in BioSci</li>
+</ul>
+<p>Next step: Compare sequence to predicted, submission process</p>
+<p class="obj">Objective: Test if &#8710;Bbs1 site was removed</p>
+<p>Miniprep Colonies</p>
+<ul>
+<li>Qiagen Set</li>
+<li>Concentrations of &#8710;Bbs1 mCherry-pSB1C3 (1-4) (ng/&mu;L): 168.0, 110.2, 175.7, 147.5</li>
+</ul>
+<p>Digest mCherry-pSB1C3 plasmid</p>
+<ul>
+<li>Standard diagnostic digest protocol used on four overnight colonies from 6/22</li>
+<li>Each colony had two digests: EcoR1 and EcoR1/Bbs1</li>
+</ul>
+<p>Run and observe gels</p>
+<ul>
+<li>Gels run for 17 minutes</li>
+<li>Order: 2-log ladder, EcoR1-cut sample 1, EcoR1 and Bbs1-cut sample 1, EcoR1-cut sample 2, EcoR1 and Bbs1-cut sample 2, EcoR1-cut sample 3, EcoR1 and Bbs1-cut sample 3, EcoR1-cut sample 4, EcoR1 and Bbs1-cut sample 4</li>
+</ul>
+<p class="obj">Objective: Insert sgRNA sequence into rCsy4-sgRNA-rCsy4 Scaffold</p>
+<p>Cut Scaffold with Bsa1 enzyme</p>
+<ul>
+<li>36 &mu;L of DNA template was cut with 5 &mu;L of enzyme in 10 &mu;L of CutSmart buffer and 49 &mu;L of water</li>
+<li>Digested for 2 hours</li>
+</ul>
+<ul>
+<li>Qiagen Set clean-up protocol performed</li>
+<li>Concentrations of rCsy4-sgRNA-rCsy4 Scaffold (ng/&mu;L): 93.2</li>
+</ul>
+<p>Anneal mCherry1 sgRNA from oligos</p>
+<ul>
+<li>crRNA-ch1-up and crRNA-ch1-dn oligos annealed using ____ protocol</li>
+</ul>
+<p>Ligate into scaffold</p>
+<ul>
+<li>Ligate annealed oligos into the cut rCsy4-sgRNA-rCsy4 scaffold using ligation protocol</li>
+</ul>
+<p>Insert Tet1 sgRNA into scaffold using SLIC</p>
+<ul>
+<li>Used standard SLIC protocol, using scaf-crRNA-Tet1-up and scaf-crRNA-Tet1-dn</li>
+</ul>
+<p>Transform into competent cells</p>
+<ul>
+<li>Used iGEM stock comp cells</li>
+<li>Plated onto LB+Cm plates</li>
+</ul>
+<p class="obj">Objective: Get pSB1C3 backbone from BBa_R0030</p>
+<p>Awaken and Transform R0030 into cells</p>
+<ul>
+<li>BBa-R0030 awakened from frozen iGEM supply</li>
+<li>Transformed onto iGEM stock competent cells and plated onto LB+Cm</li>
+</ul>
+</div>
+</div>
 <div class="day">
-<a id=""><h2></h2></a>
+<a id="jun24"><h2>June 24</h2></a>
-<div class="obj"></div>
-<div class="ppl"></div>
 <div class="lab">
-<p></p>
+<p class="obj">Objective: Check plates from 6/22/13</p>
+<p>Ligation and SLIC of sgRNA</p>
 <ul>
-<li></li>
+<li>Backbone only and experimental plates have many colonies</li>
+<li>Theories are that Bsa1 has degraded or that the plasmid self-ligated</li>
+<li>Next step: test Bsa1 </li>
+</ul>
+<p class="obj">Objective: Test Bsa1 effectiveness</p>
+<p>Digest test plasmids with Bsa1</p>
+<ul>
+<li>TAL 16C, 20A and 20B plasmids used (from last year&rsquo;s stocks in iGEM Box 3)</li>
+<li>Standard analytical restriction digest used with Bsa1 in CutSmart</li>
+<li>Left for 2 hours in 37&deg;C</li>
+</ul>
+<p>Ran diagnostic gel</p>
+<p>1. TAL 16C cut with BsaI (expected 2 fragments around 2.3 kb and one around 5.6 kb)</p>
+<p>2. TAL 20A cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)</p>
+<p>3. TAL 20B cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)</p>
+<ul>
+<li>Results: Bsa1 does cut, but fragments were not exactly expected sizes</li>
+</ul>
+<ul>
+<li>16C showed one smeary band ~ 2-3 kb plus a faint, thin band ~ 5-6kb</li>
+<li>20A showed two clear bands at ~4-5 kb and ~2-3 kb</li>
+</ul>
+<ul>
+<li>Known to have issues, may be incorrect insert</li>
+</ul>
+<ul>
+<li>20B showed two clear bands at ~6 kb and 2-3 kb</li>
+</ul>
+<p>gel picture:</p>
+<p class="obj">Objective: Attempt mCherry Bbs1 Mutation</p>
+<p>Charlie Cooper tried today.</p>
+<ul>
+<li>BsmBI digest: </li>
+</ul>
+<ul>
+<li>30uL of 159.1ng/uL pSB1C3-BBa_J06902 (mCherry) digested with BbsI in 100uL reaction &amp; NEBuffer 2.1 for 4h237C. Collected with PCR cleanup protocol and eluted in to 80uL h2o. Ran 2uL on a gel next to undigested DNA at same final concentration. Untreated plasmid runs faster than BbsI-treated, consistent with behavior of un-digested circular DNA. </li>
+<li>
+<!--https://2014.igem.org/File:6-24-14.jpg or https://static.igem.org/mediawiki/2014/a/a0/6-24-14.jpg-->
+<a href="https://static.igem.org/mediawiki/2014/a/a0/6-24-14.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/a0/6-24-14.jpg"></a>
+</li>
+<li>1: ladder</li>
+<li>2: digested plasmid</li>
+<li>3: undigested</li>
+<li>4,5 unrelated stuff for CBC</li>
+</ul>
+<ul>
+<li>CIP: </li>
+</ul>
+<ul>
+<li>Added 10uL buffer 3 and 2uL undiluted CIP. 37C for 60m. PCR cleanup again and eluted in to final volume of 35uL with conc ~76 ng/uL</li>
+</ul>
+<ul>
+<li>SLIC</li>
+</ul>
+<ul>
+<li>1 tube with 1 uL each of SLIC oligos mCherry-Bbs1-left and -rightprepared by speedVac to remove solvent. tubes placed on ice.</li>
+<li>T4 reaction set up with 2 uL backbone (0 for insert-only), buffer, bsa and water to 10uL. 0.5 uL T4 poly added and incubated at RT. at 2.5m, tube contents transferred to waiting tube with (or without for bbb-only) dried oligos. </li>
+<li>10m on ice, add 60uL comp. DH5alpha, 20m more on ice then 45s heat shock and 2m more on ice before recovering for 60m in SOC and spreading on LB+Cm and 37C overnight</li>
+</ul>
+<p class="obj">Objective: Build pDGC3 construct</p>
+<p>PCR of pLac-GFP-A-Z from pSB1C3-BBa_K741002</p>
+<ul>
+<li>4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template</li>
+</ul>
+<ul>
+<li>6/16 dilution of 6/14 miniprep</li>
+</ul>
+<ul>
+<li>Oligos pSB1C3up and GFP-A-Z-3p</li>
+<li>iGEM stock of Q5 polymerase/buffer</li>
+<li>iGEM thermocycler protocol with 30 sec extension, 65C anneal</li>
+</ul>
+<p>PCR of Asc1-mCherry-Term from pSB1C3-BBa_J06702</p>
+<ul>
+<li>4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template</li>
+</ul>
+<ul>
+<li>6/16 dilution of 6/14 miniprep</li>
+</ul>
+<ul>
+<li>Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3dn</li>
+<li>iGEM stock of Q5 polymerase/buffer</li>
+<li>iGEM thermocycler protocol with 30 sec extension, 67C anneal</li>
+</ul>
+<p>Agarose gel of PCR products:</p>
+<ul>
+<li>Successful product in lanes 2 and 4 of GFP, and 2, 3, 4 of mCherry PCR</li>
+</ul>
+<p>gel picture:</p>
+<p>PCR cleanup of GFP and mCherry amplicons</p>
+<ul>
+<li>Accidentally pooled all four tubes of GFP together</li>
+</ul>
+<ul>
+<li>Should not be a problem, may reduce yield slightly</li>
+</ul>
+<ul>
+<li>Correctly pooled 3 tubes of mCherry</li>
+<li>Final concentrations:</li>
+</ul>
+<p class="obj">Objective: Culture 16 colonies from various plates:</p>
+<p>pSB1C3-BBa_B0030</p>
+<ul>
+<li>Transformed 6/23</li>
+</ul>
+<ul>
+<li>Plate had &gt;100 colonies</li>
+</ul>
+<ul>
+<li>Cultured 1 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Next steps: use to extract pSB1C3 backbone without gel purification</li>
+</ul>
+<p>Gibson products of pDGC3 assembly</p>
+<ul>
+<li>Transformed on 6/20</li>
+<li>Backbone+Insert plate had ~75 colonies</li>
+</ul>
+<ul>
+<li>Cultured 3 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Insert-only control plate had ~200-300 colonies</li>
+</ul>
+<ul>
+<li>Cultured 2 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Backbone-only control had ~15-20 colonies</li>
+</ul>
+<ul>
+<li>Cultured 2 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Next steps: analytical digests to test if any products were successful, and why insert-only products are so numerous</li>
+</ul>
+<ul>
+<li>Insight into how to move forward with troubling construct?</li>
+</ul>
+<p>Ligation products of crRNA mCh1 into Repeat-seq scaffold</p>
+<ul>
+<li>Transformed on 6/23</li>
+<li>Backbone+Insert plate had hundreds of tiny colonies</li>
+</ul>
+<ul>
+<li>Cultured 3 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Backbone-only control had hundreds of tiny colonies</li>
+</ul>
+<ul>
+<li>Cultured 1 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Insert-only control had no colonies</li>
+<li>Next steps: Test to see if any products were successful, or which false construct caused backbone-only products</li>
+</ul>
+<p>SLIC products of crRNA Tet1 into Repeat-seq scaffold</p>
+<ul>
+<li>Transformed on 6/23</li>
+<li>Backbone+Insert plate had hundreds of tiny colonies</li>
+</ul>
+<ul>
+<li>Cultured 3 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Backbone-only control had hundreds of tiny colonies</li>
+</ul>
+<ul>
+<li>Cultured 1 in 5mL LB+Cm</li>
+</ul>
+<ul>
+<li>Insert-only control had no colonies</li>
+<li>Next steps: Test to see if any products were successful, or which false construct caused backbone-only products</li>
+</ul>
+<p class="obj">Objective: Find constitutive GFP constructs for Molecular Titration approach</p>
+<p>Transformed four biobricks in pSB1C3 from iGEM kit plates</p>
+<ul>
+<li>Plate 1-5I:BBa_K608008</li>
+</ul>
+<ul>
+<li>&ldquo;strong promoter/medium RBS + GFP&rdquo;</li>
+</ul>
+<ul>
+<li>Plate 1-5K:BBa_K608010</li>
+</ul>
+<ul>
+<li>&ldquo;medium promoter/strong RBS + GFP&rdquo;</li>
+</ul>
+<ul>
+<li>Plate 1-5M:BBa_K608011</li>
+</ul>
+<ul>
+<li>&ldquo;medium promoter/medium RBS + GFP&rdquo;</li>
+</ul>
+<ul>
+<li>Plate 1-5O:BBa_K608012</li>
+</ul>
+<ul>
+<li>&ldquo;medium promoter/weak RBS + GFP&rdquo;</li>
+</ul>
+<ul>
+<li>Transformed by heat shock using iGEM CCEC stocks</li>
+</ul>
+<ul>
+<li>Plated on LB+Cm</li>
 </ul>
 </div>
 </div>
-<!-- Comment here -->
+<div class="day">
-<!-- To insert a picture, the command is <img src=""> -->
+<a id="jun25"><h2>June 25</h2></a>
+<div class="lab">
+<p class="obj">Objective: Check plates from 6/24/13</p>
+<p>Check &#8710;Bbs1 Plates</p>
+<ul>
+<li>Backbone only and experimental plates have many colonies, leading to possibility that CIP did not function</li>
+</ul>
+<p class="obj">Objective: Check CIP still functions</p>
+<p>Digest plasmid with EcoRI</p>
+<ul>
+<li>pSB1C3-J06702 &#8710;Bbs1 plasmids 8 and 9 were used</li>
+<li>Standard ligation prep digest preformed on 32 uL of template in CutSmart buffer</li>
+<li>Digest cleaned with Qiagen PCR clean-up into 30uL of EB</li>
+<li>Concentration of &#8710;Bbs1 8 was 99.2 ng/uL and 9 was 145.6 ng/uL.</li>
+</ul>
+<p>Treat experimental group with Calf Intestinal Phosphotase</p>
+<ul>
+<li>15 uL of the cleaned DNA included in standard CIP protocol</li>
+<li>CIP cleaned with Qiagen PCR clean-up into 15 uL of EB</li>
+<li>Concentration of &#8710;Bbs1 8 was 32.6 ng/uL and 9 was 62.2 ng/uL.</li>
+</ul>
+<p>Ligate plasmids with T4</p>
+<ul>
+<li>Ligation protocol used triple amounts of all compounds as standard 100 ng ligations, producing 300 ng of ligated DNA for &#8710;Bbs1-8, &#8710;Bbs1-8-CIP, &#8710;Bbs1-9 and &#8710;Bbs1-9-CIP in 60 uL</li>
+<li>40 uL was cleaned with a Qiagen PCR clean-up into 8.5 uL of EB</li>
+<li>20 uL was left to transform into cells</li>
+</ul>
+<p>Digest with EcoRV and analyze digestion</p>
+<ul>
+<li>8.5 uL of DNA, since concentrations are so low, were used in a restriction digest with 1 uL of CutSmart and 0.5 uL of EcoRV</li>
+<li>Ran on gel to test digest</li>
+<li>Order: 2-log, &#8710;Bbs1-8, &#8710;Bbs1-8-CIP, &#8710;Bbs1-9, &#8710;Bbs1-9-CIP</li>
+</ul>
+<p>Transform DNA into cells to see rate of uptake</p>
+<ul>
+<li>Transformed into iGEM comp cells and plated onto LB+Cm</li>
+</ul>
+<p class="obj">Objective: Build crRNAs in Repeat-seq scaffold</p>
+<p>Miniprep crRNA cultures and BO controls</p>
+<ul>
+<li>3 copies crRNA-Tet1 and one BO control</li>
+</ul>
+<ul>
+<li>Concentrations in 50 uL (1,2,3,BO):</li>
+</ul>
+<ul>
+<li>68, 124.7, 71.4, 89.2 ng/uL</li>
+</ul>
+<ul>
+<li>3 copies crRNA-mCh1 and one BO control</li>
+</ul>
+<ul>
+<li>Concentrations in 50 uL (1,2,3,BO): </li>
+</ul>
+<ul>
+<li>55.8, 51.3, 60.6, 65.1 ng/uL</li>
+</ul>
+<ul>
+<li>Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)</li>
+</ul>
+<p>Analytical digest of crRNA-Tet1 minipreps</p>
+<ul>
+<li>To determine if minipreps still digestible (if in glycerol, may not be)</li>
+<li>4 tubes digested with EcoRI-HF/EcoRV-HF in cutsmart buffer for 2 hrs</li>
+</ul>
+<p>Agarose gel of digest results:</p>
+<p>1. crRNA-Tet1-1 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)</p>
+<p>2. crRNA-Tet1-2 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)</p>
+<p>3. crRNA-Tet1-3 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)</p>
+<p>4. crRNA-Tet1-BO in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)</p>
+<ul>
+<li>Results: expected bands present, minipreps still useful in digests</li>
+</ul>
+<p>Analytical digest of crRNA minipreps</p>
+<ul>
+<li>To test whether new insert is present</li>
+<li>8 tubes digested with BsaI/NcoI in cutsmart buffer for 2 hrs</li>
+<li>Frozen in iGEM freezer--need to run on gel</li>
+</ul>
+<p class="obj">Objective: Build pDGC3</p>
+<p>MIniprep pDGC3 cultures, BO controls, and IO controls</p>
+<ul>
+<li>3 copies pDGC3, 2 copies BO control, 2 copies IO control</li>
+</ul>
+<ul>
+<li>Concentrations in 50 uL (1,2,3,BO-1,BO-2,IO-1,IO-2):</li>
+</ul>
+<ul>
+<li>55.6, 66.9, 54.1, 32.1, 36.2, 55.5, 58.7 ng/uL</li>
+</ul>
+<ul>
+<li>Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)</li>
+</ul>
+<p>Analytical digest of minipreps</p>
+<ul>
+<li>To test whether GFP and/or mCherry are present</li>
+<li>Two digest each of 7 samples, NdeI/EcoRV-HF and BstEII-HF/EcoRV-HF</li>
+<li>All in cutsmart buffer for 2 hrs</li>
+<li>Frozen in iGEM freezer--need to run on gel</li>
+</ul>
+<p class="obj">Objective: Obtain pSB1C3 backbone without gel purification</p>
+<p>Miniprep pSB1C3-B0030 culture</p>
+<ul>
+<li>Concentration 34 ng/uL (eluted in 50 uL)</li>
+<li>Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)</li>
+</ul>
+<p>Prep-scale digest of pSB1C3-B0030 to obtain backbone</p>
+<ul>
+<li>Enzymes SpeI-HF/EcoRI-HF in cutsmart buffer</li>
+<li>42 uL DNA in 100 uL reaction for 4 hrs</li>
+<li>Note: product frozen in iGEM cloning project box overnight </li>
+<li>Need to run on gel to confirm and PCR clean to use</li>
+</ul>
+<p class="obj">Objective: Culture colonies containing constitutive GFP for molecular titration approach</p>
+<ul>
+<li>pSB1C3-B0030(as a control with no flourescence)</li>
+<li>pSB1C3-K608008&ldquo;strong promoter/medium RBS + GFP&rdquo;</li>
+<li>pSB1C3-K608010&ldquo;medium promoter/strong RBS + GFP&rdquo;</li>
+<li>pSB1C3-K608011&ldquo;medium promoter/medium RBS + GFP&rdquo;</li>
+<li>pSB1C3-K608012&ldquo;medium promoter/weak RBS + GFP&rdquo;</li>
+<li>To flow tomorrow and save glycerol stocks</li>
+</ul>
 </div>
+</div>
+<div class="day">
+<a id="jun26"><h2>June 26</h2></a>
+<div class="lab">
+<p class="obj">Objective: Check plates from 6/25/13 to confirm CIP activity</p>
+<p>Check &#8710;Bbs1-8 and 9 Plates</p>
+<ul>
+<li>Still many colonies on CIP treated plates, but much fewer than non-CIP treated plates</li>
+<li>This leads to the conclusion that CIP still functions, but is not completely effective, leading to possible expression of self-ligation in CIP treated &#8710;Bbs1 experiments</li>
+</ul>
+<p class="obj">Objective: Dephosphorylate Bbs1 cut pSB1C3-J06702 (mCherry)</p>
+<p>Run dephosphorylation</p>
+<ul>
+<li>3uL of Cutsmart and 1.5 uL of CIP added to 25 uL of mCherry plasmid, set in 37C for 45 minutes</li>
+<li>DNA cleaned with Qiagen PCR Clean-up</li>
+</ul>
+<p class="obj">Objective: Analyze the constitutive GFP expression of reporters using flow cytometry</p>
+<p>Save stocks of Constitutive GFP plasmids</p>
+<ul>
+<li>K608008, K608010, K608011, K608012</li>
+</ul>
+<p>Flow Cytometry</p>
+<ul>
+<li>FSC log 3, 350V</li>
+<li>SSC log 3, 350V</li>
+<li>B1 log 5, 350V</li>
+<li>Order of samples:</li>
+</ul>
+<p>1. B0030 (control)</p>
+<p>2. K608008</p>
+<p>3. K608010</p>
+<p>4. K608011</p>
+<p>5. K608012</p>
+<p>Results: (analyzed 6/30/14)</p><a href="#" name="9c0305aa5f2167c62a3b4b16e84255280c661385"></a><a href="#" name="2"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td></td><td><p>E. coli/Simple peak GFP | Mean (B1-A)</p></td><td><p>Fold Change from control</p></td></tr><tr><td><p>pSB1C3-B0030 (control)</p></td><td><p>0.29</p></td><td><p>1</p></td></tr><tr><td><p>pSB1C3-K608008</p></td><td><p>0.3</p></td><td><p>1.03</p></td></tr><tr><td><p>pSB1C3-K608010</p></td><td><p>201</p></td><td><p>693.10</p></td></tr><tr><td><p>pSB1C3-K608011</p></td><td><p>58.2</p></td><td><p>200.69</p></td></tr><tr><td><p>pSB1C3-K608012</p></td><td><p>118</p></td><td><p>406.90</p></td></tr></tbody></table><p>Notes: </p>
+<ul>
+<li>K608008 showed no fluorescence--could be transformation problem or part problem. </li>
+<li>K608010 had messier data for FSC/SSC and for GFP (B-1). Despite having the highest mean, this is not as reliable of a sample.</li>
+<li>K608012 had the highest reliable mean, and should be used for at least the initial molecular titration experiments. K608011 is also useful if we need a lower maximum fluorescence level. </li>
+</ul>
+</div>
+</div>
+<div class="day">
+<a id="jun30"><h2>June 30 </h2></a>
+<div class="lab">
+<p class="obj">Objective: Insert crRNAs targeting GFP into pdCas9 and Repeat scaffold</p>
+<p>Prep-scale Digest of pdCas9</p>
+<ul>
+<li>BsaI in Cutsmart buffer</li>
+<li>30 uL template in 100 uL reaction</li>
+<li>4 hrs at 37C</li>
+</ul>
+<p>Anneal crRNA inserts</p>
+<ul>
+<li>3 inserts, each annealed with 2 oligos</li>
+</ul>
+<ul>
+<li>crRNAs GFP1, GFP2, and GFP3</li>
+</ul>
+<ul>
+<li>5uL each oligo in ~boiling water bath, cooled at rt to rt </li>
+</ul>
+<p>Ligation of crRNAs into BsaI site in pdCas9 and Repeat scaffold</p>
+<ul>
+<li>6 reactions plus 5 controls</li>
+<li>pdCas9 with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)</li>
+</ul>
+<ul>
+<li>backbone cut with BsaI (6/30), 100ng=3.5uL</li>
+</ul>
+<ul>
+<li>pSB1C3-Repeat-scaffold (pDGC1) with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)</li>
+</ul>
+<ul>
+<li>backbone cut with BsaI (6/23), 100ng=1uL</li>
+</ul>
+<ul>
+<li>BO controls for pdCas9 and Repeat-scaffold</li>
+<li>IO controls for all three crRNA inserts</li>
+<li>10 uL rxns transformed into DH5alphas (iGEM&rsquo;s CCEC), plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Build pDGC3</p>
+<p>Gibson assembly of pSB1C3, GFP, and mCherry</p>
+<ul>
+<li>Backbone cut with EcoRI/SpeI on 6/16, 100ng=1.5uL</li>
+<li>GFP PCR treated with DpnI on 6/26</li>
+<li>mCherry PCR treated with DpnI on 6/26</li>
+<li>Two copies, one with 100ng backbone, one with 50ng backbone</li>
+</ul>
+<ul>
+<li>1xMolar and 2xMolar inserts (50ng each)</li>
+</ul>
+<ul>
+<li>BO and IO controls</li>
+<li>Using Mert&rsquo;s new DIY Gibson mix (15uL into 20uL)</li>
+<li>Transformed into DH5alphas (iGEM&rsquo;s CCEC), plated on LB+Cm</li>
+</ul>
+<p class="obj">Objective: Remove BbsI site from mCherry</p>
+<p>SLIC to remove BbsI site from pSB1C3-J06702</p>
+<ul>
+<li>Backbone pSB1C3-J06702 super-treated with CIP on 6/26, 100ng=2.5uL</li>
+<li>Oligos mCherry-BbsI-left and mCherry-BbsI-right</li>
+<li>10 uL rxns transformed into DH5alphas (iGEM&rsquo;s CCEC), plated on LB+Cm</li>
+</ul>
+</div>
+</div>
+<!-- or -->
+<a href="" target="_blank"><img src=""></a>
+<!--
+Gel picture links from Matt Farnitano
+GEL 6_10_14_AJC.png
+GEL 6-11-14_MCF.png
+GEL 6_12_14_AJC.png
+GEL 6_12_14_3-40_AJC.png
+GEL 6_13_14.png
+GEL 6-13-14__2.png
+GEL 6-13-14_MCF_bbs1.png
+GEL 6-13-14.png
+GEL 6-16-14_AJC.png
+GEL 6-16-14_MCF.png
+GEL 6-16-14_2.png
+GEL 6-16-14_4.png
+GEL 6-18-14_MCF.png
+GEL 6-20-14_MCF.png
+GEL MCF_6-17-14.png
+GEL MCF_6-18-14.png
+GEL Mcf_6-19-14.png
+GEL 6-23-14.png
+GEL MCF_6-24-14_2.png
+GEL MCF_6-24-14.png
+GEL MCF 6-25-14.png
+GEL 6-25-14_AJC.png
+GEL 6-26-14_MCF.png
+-->
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