Team:Duke/Notebook/July

Miniprep 40 cultures of potential crRNA inserts in pdCas9

• Labeled with three numbers
• Date of transformation - crRNA-GFPx - sample number

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

• Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

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

Made and ran 1.5% agarose/TBE gels

• To differentiate between small band differences
• Gel 1:
• 1. 2-log ladder
• 2. pdCas9
• 3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
• 13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
• 23. pdCas9
• 24. 2-log ladder
• Gel 2:
• 1. 2-log ladder
• 2. pdCas9
• 3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
• 13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
• 23. pdCas9
• 24. 2-log ladder
• Expected results:
• pdCas9 and successful inserts:
• 3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band
• unsuccessful recircularizations:
• 3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band
• Results:
• Gel 1 appears to only be recircularized
• Gel 2 has some promising inserts:
• GFP1 samples 1,2, and 3 from 7/7 transformation
• GFP2 samples 7,8, and 9 from 7/7 transformation
• Glycerol stocks frozen for these six samples
• Next Steps:
• Run digest with BsaI to confirm that plasmids are not uncut pdCas9
• Sequence to confirm presence of insert (using oligos ordered 7/8/14)

Miniprep 3 new backbones

• pSB6A1-J04450 (3 copies)
• concentrations 437.6, 483.9, and 450.1 ng/uL
• pSB3K3-I20260 (3 copies)
• concentrations 114.6, 120.0, and 97.3 ng/uL
• pSB4K5-J04450 (3 copies)
• concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

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

Agarose gel extraction of 3 backbones

• Gel 1, Left: pSB6A1
• Gel 1, Right: pSB3K3
• Gel 2, Left: pSB4K5
• All 3 lanes had 2 bands as expected
• upper band (backbone) in 3K3 had messy trail behind it (not extracted)
• All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
• top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

• pSB1C3-K608012
• In order to switch into pSB6A1

Transformation from iGEM distribution kit plates

• Plate 2-6D: pSB1C3-BBa_R0011
• Engineered Lac promoter (not affected by glucose)
• Plate 3-15O: pSB1C3-BBa_I13500
• Strong RBS-GFP
• Plated on LB+Cm

Gel cleanup of 3 backbones with Zymoclean prep kit

• Some tubes had to be divided into two samples for cleanup
• Final elution was 20 uL each into 2-3 tubes per backbone
• Nanodrops looked unclean: Most had high peak around 220-240 nm
• pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL
• Peak ~220, then hump ~260
• pSB6A1: 270 mg gel = 220 ng/uL
• Peak, then hump as before
• pSB3K3: 320 mg gel = 53.9 ng/uL
• No clear sign of DNA in graph
• pSB3K3: 230 mg gel = 20 ng/uL
• No DNA
• pSB3K3: 130 mg gel = 72 ng/uL
• Possibly DNA, best option but may not be useful
• pSB4K5: 290 mg gel = 43.4 ng/uL
• No clear sign of DNA in graph
• pSB4K5: 320 mg gel = 117.3 ng/uL
• Possibly DNA, best option but may not be useful

Culture 3 colonies of pSB1C3-K608012

• In order to switch into pSB6A1

Analytical Digest of promising crRNA inserts

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

Agarose gel of digest results:

• 1. pdCas9
• 2-4. pdCas9-crRNA-GFP1, samples 1,2,3
• 5-7. pdCas9-crRNA-GFP2, samples 7,8,9
• Expected:
• 2 bands at 4.2 and 5.1 kb in pdCas9
• 1 band at 9.3 kb in successful plasmids with inserts
• Results:
• pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band
• Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.

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

Culture pSB1C3-I13521 (pTet-RFP)

• For flow
• In aTc and non-aTc

Miniprep 3 copies of pSB1C3-K608012

• concentrations 361, 324, 362 ng/uL

Prep-scale digest of pSB1C3-K608012

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

Gel extraction and cleanup to isolate K608012

• Cut lower band
• Zymoclean prep kit
• Each of 2 bands divided into two tubes during cleanup
• 1A: 280 mg =
• 1B: 240 mg =
• 2A: 200 mg =
• 2B: 200 mg = 62.5 ng/uL (20 uL)
• Graphs on nanodrop did not look ideal: peak lower than usual

Miniprep pSB1C3-R0011 and pSB1C3-I13500

• 3 copies each
• Concentrations
• pSB1C3-R0011: 181, 229, 194 ng/uL
• pSB1C3-I13500: 321.5, 228, 300.4 ng/uL

Prep scale digests of two tubes from each miniprep

• pSB1C3-R0011 with SpeI/PstI
• no extraction necessary
• pSB1C3-I13521 with XbaI/PstI
• to extract I13521 insert
• 100 uL total reaction per tube
• 2-3 hrs at 37C

Agarose gel of pSB1C3-I13500 XbaI/PstI digests

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

Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests

• Single band appears correct
• see below for gel picture

PCR cleanup of pSB1C3-R0011 SpeI/PstI digests

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

Miniprep pdCas9

• concentration 167.4 ng/uL

Prep-scale digest of pSB1C3-R0011

• 1 miniprep with XbaI/PstI
• To extract pSB1C3 backbone

Agarose gel of pSB1C3-R0011 XbaI/PstI digest

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

PCR of dCas9-tracrRNA and anti-tracrRNA

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

Agarose gel of PCR (and digest) results:

• Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
• Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
• Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests
• Results:
• dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes
• tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension.
• tracrRNA PCR should be done with shorter extension time

PCR cleanup of dCas9-tracrRNA PCR

• Concentration >200 ng/uL

Analytical digest of dCas9

• One tube with BsaI/EcoRI
• One tube with XbaI/EcoRI
• XbaI from tube with exp. 5/14
• One tube with XbaI/EcoRI
• XbaI from tube with exp. 2/15

Agarose gel of digest:

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

Sequencing Order #1608

• BigDye reaction with BD buffer 1.1
• pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9up1
• pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9up1
• pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9up1
• pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9up1
• pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9up1
• pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9up1
• pdCas9-crRNA_GFP3 sample 3 w/ pdCas9up1
• pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9dn1
• pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9dn1
• pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9dn1
• pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9dn1
• pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9dn1
• pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9dn1
• pdCas9-crRNA_GFP3 sample 3 w/ pdCas9dn1

Results:

• pdCas9-crRNA_GFP1 use sample 7-1
• pdCas9-crRNA_GFP2 use sample 7-9
• pdCas9-crRNA_GFP3 need to find more colonies

Prep scale digest of pSB1C3-I13500 with XbaI/PstI

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

Agarose gel extraction and cleanup of pSB1C3 and I13500

• Top band (~2kb): pSB1C3
• Bottom band (~1kb): I13500
• Zymoclean prep kit
• Concentrations:
• pSB1C3: 319 ng/uL in 20 uL (dirty)
• I13500: 91.2 ng/uL in 20 uL (dirty)

Ligation of pSB1C3-R0011 and I13500

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

Ligation of pSB6A1 and K608012

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

Prep scale digests

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

PCR cleanup of dCas9-tracr digest

• Qiagen miniprep kit
• Concentration 38.1 ng/uL in 30 uL

Agarose gel extraction and cleanup of pSB1C3

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

Ligation of pSB1C3 and dCas9-tracrRNA PCR

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

Prep-scale digest of pSB1C3-R0040

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

PCR anti-tracrRNA from pdCas9

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

Agarose gel of anti-tracrRNA PCR

• Results: only 1.0 uL template lane appeared to amplify
• Need to make more using this template concentration

PCR anti-tracrRNA from pdCas9

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

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

• Lanes 1-8: anti-tracrRNA PCR
• Lane 9: R0040 digest
• Results:
• PCR appears to work in all lanes (band size ~0.1 kb)
• R0040 digest single band appears correct

PCR cleanup of anti-tracrRNA PCR and pSB1C3-R0040

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

Digest of anti-tracrRNA PCR

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

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

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

Ligation of anti-tracrRNA and pSB1C3-R0040

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

Plate results:

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

Culture colonies

• 4 copies of potential pSB1C3-dCas9-tracrRNA construct

Digest of dCas9-tracrRNA PCR and heat inactivation

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

PCR Cleanup of pSB1C3 digest

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

Ligation of dCas9-tracrRNA PCR and pSB1C3

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

Plate results:

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

Digest of pSB1C3-R0011

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

PCR Cleanup of pSB1C3-R0011 digest and I13500 digest

• pSB1C3-R0011 concentration 175 ng/uL
• I13500 (from previous digestion and extraction)
• 2.3 ug original yields 124 ng/uL in 20 uL “superclean”

Ligation of I13500 and pSB1C3-R0011

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

Plate results:

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

Cultured colonies

• 2 colonies of potential pSB6A1-K608012 construct

PCR Cleanup of pSB6A1 digest and K608012 digest

• pSB6A1, 2 tubes gel extracted and cleaned
• 10 ug original yields 203 ng/uL in 30 uL
• K608012, 3 tubes gel extracted and cleaned
• 4 ug original yields 73.0 ng/uL in 30 uL

Ligation of K608012 and pSB6A1

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

Plate results:

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

Cultured colonies

• 4 copies of potential pSB1C3-R0040-anti-tracrRNA

Plate results:

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

Minipreps

• 4 copies of potential pSB1C3-dCas9-tracrRNA construct
• From 7/14 ligation
• concentrations: 537.2, 45.4, 79.1, and 71.5 ng/uL
• Only one has normal concentration, others very low

Analytical digest of pSB1C3-dCas9-tracrRNA constructs:

• 4 copies with NheI/XbaI

Agarose gel of digests:

• Lanes 1-2: pSB6A1-K608012 with MfeI/XbaI (expected 4.1, 0.6 kb bands)
• Lanes 3-6: pSB1C3-dCas9-tracrRNA with NheI/XbaI (expected 5.0, 1.5 kb bands)
• Results: gel was fuzzy and unclear
• lanes 2,3,5,6 clearly incorrect: 1 band ~2.0 kb
• lane 1 had one fuzzy band around 4-5 kb (could be incomplete digest)
• lane 4 had band around 5 kb with smear above (could be incomplete digest)

New analytical digest of pSB1C3-dCas9-tracrRNA construct

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

Agarose gel of digests

• DETAILS

Plate results:

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

Cultured colonies

• 4 copies of potential pSB1C3-R0011-I13500 construct

Plate results:

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

Colony PCR of pdCas9-GFP3 plate

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

Agarose gel of colony PCR

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

Cultured colonies

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