Team:Duke/Notebook/July
From 2014.igem.org
July 9
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
July 10
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
July 11
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
July 14
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
July 15
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
July 16
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
July 17
Minipreps
- 4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation
- 4 copies of pSB1C3-R0011-I13500 from 7/15 ligation
Analytical digest of minipreps
- pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI
- pSB1C3-R0011-I13500 with MfeI/NdeI/SacI
Agarose gel of digest results:
- Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, <0.1 kb bands)
- Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)
- Results:
- R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly
- R0011-I13500 does not look correct (only one band at 2.0 kb)
Colony PCR on pSB1C3-dCas9-tracrRNA plate
- Ligation plate from 7/15/14
- 15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC
- oligos SB1C3-up1 and SB1C3-dn1
- annealed at 65C, extension 2 mins
Agarose gel of colony PCR
- results: primer-bands only (no successes)
Culture colonies
- 2 copies of pSB6A1-J04450 (from 6-9-14 plate)
- 2 copies of pSB1C3-K608012
July 18
Objective: Switch dCas9-tracrRNA into pSB1C3
PCR of dCas9-tracrRNA from pdCas9
- 8 tubes, each with 1 uL pdCas9 template at 1 ng/uL
- oligos dCas9tracr-up and dCas9-tracr-dn
- 2 min extension, 64C anneal
Agarose gel of PCR product
- All 8 tubes look successful (band ~4 kb)
PCR cleanup of dCas9-tracrRNA
- Qiagen kit
- Into 2 tubes, 30 uL each
Prep-scale digest of PCR product
- with PstI/XbaI/DpnI
- in 100 uL each for 3 hrs at 37C
Treat pSB1C3 digest with CIP
- 1 hr at 37C
Objective: Switch K608012 into pSB6A1
Miniprep pSB1C3-K608012 and pSB6A1-J04450
- 1 copy each (the second copy of each did not grow in culture)
- concentrations:
- pSB1C3-K608012: 344 ng/uL
- pSB6A1-J04450: 421.5 ng/uL
Prep-scale digest of K608012 and pSB6A1
- 50 uL DNA in 100 uL reaction
- Both with EcoRI and PstI
- 3 hrs at 37C
CIP treatment of pSB6A1 and heat-inactivation
- 2.5 uL CIP added to digest tube
- Both digests returned to 37C for 1 hr
- Then 30 mins at 80C to heat-inactivate
Gel extraction of pSB6A1 and K608012
- Gel had no EtBr added, and was discarded (product was lost)
July 20
Objective: culture colonies for various purposes
Cultured colonies
- 4 copies pSB6A1-J04450
- 2 from older plate, 2 from newer
- 4 copies pSB1C3-K608012
- 2 from older plate, 2 from newer
- 4 copies pSB1C3-B0030
- To obtain pSB1C3 without gel extraction
- 4 copies pSB1C3-R0011
- 4 copies pSB1C3-I13500
July 21
Objective: Sequence to confirm constructs
Sequencing reaction and Order #1634
- 1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1
- 5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1
- 9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1
- 11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1
- Big Dye, buffer version 1.1
Order 1634 Results:
- pdCas9-GFP3 copies 3, 6, and 8 are correct
- copy 5 has a single mutation
- anti-tracrRNA: both copies are correct
- copy 1 was a cleaner, more reliable
Objective: Create pSB1C3-R0011-I13500
Minipreps
- Qiagen kit
- 4 tubes of pSB1C3-R0011
- concentrations 255.7, 169.2, 204.8, 151.2 ng/uL
- 4 tubes of pSB1C3-I13500
- concentrations 148.9, 214.2, 260.7, 432.5
Prep scale digests of pSB1C3-R0011 and I13500
- pSB1C3-R0011 with SpeI/PstI, samples 1,3
- pSB1C3-I13500 with XbaI/PstI, samples 1,3
- 50 uL DNA in 100 uL each reaction, 2 tubes per construct
- 3 hrs at 37C
- Added CIP (2.5 uL) to pSB1C3-R0011 reaction
- 1 more hr for both at 37C
- Heat inactivation of both reactions: 30 mins at 80C
Gel extraction of I13500
- extraction gel 1, lanes 1 and 2
- Zymoclean prep kit
- sample 1 (lane 1): 300 mg gel yields
- sample 3 (lane 2): 550 mg gel yields
PCR cleanup of pSB1C3-R0011
- Qiagen kit
- Two tubes of digest combined into one tube product
- Concentration
Ligation of pSB1C3-R0011 and I13500
- Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL
- New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL
- Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL
- New insert (I13500 extracted 7/21): 300 ng = 3 uL
- Ligations numbered 1-8:
- 1. OB+OI
- 2. OB+NI
- 3. NB+OI
- 4. NB+NI
- 5. OB control
- 6. OI control
- 7. NB control
- 8. NI control
- 1 hr at rt
- Transformed via heat shock into DH5alphas, plated on LB+Cm
Objective: Switch dCas9-tracrRNA into pSB1C3
Minipreps
- Qiagen kit
- 4 tubes of pSB1C3-B0030
- to obtain pSB1C3 without gel extraction
- concentrations 116.8, 99.9, 115.3, 145.1 ng/uL
PCR cleanup of dCas9-tracrRNA PCR
- Product digested 7/18 with XbaI/PstI/DpnI
- Concentration 80.5 ng/uL in 30 uL
- Heat inactivation of cleaned product, 30 mins at 80C
Prep scale digest of pSB1C3-B0030
- With XbaI/PstI, samples 1,3
- 50 uL DNA in 100 uL each reaction, 2 tubes
- 3 hrs at 37C
- Added CIP (2.5 uL)
- 1 more hr for both at 37C
- Heat inactivation: 30 mins at 80C
PCR cleanup of pSB1C3
- B0030 should not be retained
- Qiagen kit
- Two tubes of digest combined into one tube product
- Concentration
Ligation of pSB1C3 and dCas9-tracrRNA
- Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL
- Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL
- New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL
- Ligations numbered 9-13:
- 9. B+OI
- 10. B+NI
- 11. B control
- 12. OI control
- 13. NI control
- 1 hr at rt
- Transformed via heat shock into DH5alphas, plated on LB+Cm
Objective: Switch K608012 into pSB6A1
Minipreps
- Qiagen kit
- 4 tubes of pSB6A1-J04450
- concentrations 189.3, 432.5, 688.7, 487.3 ng/uL
- 4 tubes of pSB1C3-K608012
- concentrations 68.8, 91.6, 346.1, ng/uL
Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012
- pSB6A1-J04450 with XbaI/SpeI, samples 1,3
- pSB1C3-K608012 with EcoRI/PstI, samples 1,3
- 50 uL DNA in 100 uL each reaction, 2 tubes per construct
- 3 hrs at 37C
- Added CIP (2.5 uL) to pSB6A1-J04450 reaction
- 1 more hr for both at 37C
- Heat inactivation of both reactions: 30 mins at 80C
Gel extraction of pSB6A1 and K608012
- K608012: gel 1, lane 3 and gel 2, lane 1
- pSB6A1: gel 2, lanes 2 and 3
- Zymoclean prep kit
- K608012 sample 1 (lane 1-3): 300 mg gel yields
- K608012 sample 3 (lane 2-1): 550 mg gel yields
- pSB6A1 sample 1 (lane 2-2):
- pSB6A1 sample 3 (lane 2-3):
Ligation of pSB6A1 and K608012
- Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL
- Old insert (K608012 extracted 7/15): 300 ng = 4 uL
- Ligations numbered 14-16:
- 14. B+I
- 15. B control
- 16. I control
- 1 hr at rt
- Transformed via heat shock into DH5alphas, plated on LB+Amp
Gibson of pSB6A1 and K608012
- New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
- New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
- Numbered 17-19:
- 17. B+I
- 18. B control
- 19. I control
- 15 uL Mert’s DIY Gibson mix
- 1 hr at 60C
- Transformed via heat shock into DH5alphas, plated on LB+Amp
SLIC of pSB6A1 and K608012
- New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
- New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
- Numbered 20-22:
- 20. B+I
- 21. B control
- 22. I control
- 2.5 mins at rt, then 10 mins at 4C
- Transformed via heat shock into DH5alphas, plated on LB+Amp
July 22
Objective: Create pSB1C3-R0011-I13500
Plate results:
- All four experimental plates had lots of colonies
- both Backbone-only controls had similar colony numbers to experimentals
- Insert-only controls had no colonies
Colony PCR
- 4 copies each of 4 experimental plates = 16 colonies
- Oligos SB1C3-up and SB1C3-dn with Taq polymerase
- 66C anneal temp, 2 min extension time
Agarose gel of colony PCR results
- 1-4: OB+OI pSB1C3-R0011-I13500
- 5-8: OB+NI pSB1C3-R0011-I13500
- 9-12: NB+OI pSB1C3-R0011-I13500
- 13-16: NB+NI pSB1C3-R0011-I13500
- 17-20: B+OI pSB1C3-dCas9-tracrRNA
- 21-24: B+NI pSB1C3-dCas9-tracrRNA
- Results: no successful bands on any colonies
- Just primer-dimers or small amplified bands?
Objective: Switch dCas9-tracrRNA into pSB1C3
Plate results:
- Both experimental plates had lots of colonies
- Backbone-only controls had similar colony numbers to experimentals
- Insert-only controls had no colonies
Colony PCR
- 4 copies each of 2 experimental plates = 8 colonies
- Oligos SB1C3-up and SB1C3-dn with Taq polymerase
- 66C anneal temp, 2 min extension time
Agarose gel of colony PCR results
- see above for gel order
- Results: no successful bands on any colonies
- Just primer-dimers or small amplified bands?
Objective: Switch K608012 into pSB6A1
Plate results:
- No colonies on Gibson or SLIC plates
- 3 colonies on Ligation plate
- A few colonies on controls, but not many
Cultured colonies
- 3 colonies of potential pSB6A1-K608012 from Ligation plate
- in LB+Amp
Ligation of K608012 and pSB6A1
- Same constructs as 7/21 ligation
- This time let recover in SOC for 2 hrs instead of 1 hr
- Plated on LB+Amp
Objective: Try putting K608012 into pSB1A2 instead
Transformation from kit plate
- Plate 4-1N
- pSB1A2-B0034
- Plated on LB+Amp
July 23
Objective: Switch K608012 into pSB6A1
Miniprep 3 copies of potential pSB6A1-K608012
- Qiagen kit
- Saved stocks in 15% glycerol
Analytical digest of minipreps
- With NdeI in Cutsmart, 10 uL final volume
Agarose gel of analytical digest
- Results:
- Copies 1 and 2 are strange and incorrect
- Copy 3 looks correct
- Need to check with more digests
Analytical digest to check promising sample
- Copy 3 in 3 different reactions
- EcoRI/SpeI
- EcoRI/MfeI
- NcoI/PvuI
- Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI
- Control: pSB1C3-K608012 with MfeI/NcoI
Agarose gel of analytical digest
- 1. Ligation 3 with EcoRI/SpeI
- 2. Ligation 3 with EcoRI/MfeI
- 3. Ligation 3 with NcoI/PvuI
- 4. pSB6A1-J04450 with EcoRI/SpeI/PvuI
- 5. pSB1C3-K608012 with MfeI/NcoI
- Results:
- Sample looks good
- Incorrect samples 1 and 2 glycerol stocks thrown out
Objective: Switch R0040-anti-tracrRNA into pSB4K5
Treat pSB4K5 with CIP
- 1.25 uL CIP in 25 uL reaction
- pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9
Prep-scale digest to obtain R0040-anti-tracrRNA
- pSB1C3-R0040-anti-tracrRNA
- with EcoRI/SpeI
Gel extraction and cleanup of R0040-anti-tracrRNA
- Extracted smaller band
- Attempted to clean up using Qiagen prep kit
- dissolved with Zymoclean ADB
- Then followed Qiagen PCR cleanup protocol
- 510 mg gel yielded little or no DNA
Objective: Create pSB1C3-R0011-I13500
Colony PCR
- 8 colonies from 7/21 ligation plates
- Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011
Agarose gel of colony PCR results
- No successes
- Controls did not work properly either
- colony PCR does not seem to be effective--need to just pick colonies
Culture colonies
- 8 colonies of potential pSB1C3-R0011-I13500
- From 7/21 ligation plates (colonies from today’s colony PCR)
Objective: Measurement interlab study
Transformations from kit plates
- Plate 1-20K
- pSB1C3-K823005
- Plate 1-22I
- pSB1C3-K823012
- Plate 2-24B
- pSB1C3-E0240
July 24
Objective: Create pSB1C3-R0011-I13500
Miniprep 8 colonies of potential pSB1C3-R0011-I13500
- Qiagen kit
Analytical digest of minipreps
- 8 copies with NcoI
- 8 copies with EcoRI/SpeI
Agarose gel of digest results
- 1-8: copies 1-8 with NcoI
- 9-16: copies 1-8 with EcoRI/SpeI
- Results: no successes, insert appears to be missing
- Unsure whether incorrect colonies are pSB1C3 or pSB1C3-R0011
Agarose gel of digest results
- Thicker gel, longer run time, more DNA
- from same digest tubes
- Results:
- no small band on EcoRI/SpeI cut and no difference in larger band for the two cuts, indicating that R0011 is not present
Ligation of pSB1C3-R0011 and I13500
- Used lab stock of T4 Ligase and buffer rather than iGEM stock
- 100 ng pSB1C3-R0011 = 2.5 uL
- 300 ng I13500 = 3 uL
- 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
- BO and IO controls for each time
- Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)
Objective: Switch dCas9-tracrRNA into pSB1C3
Ligation of pSB1C3 and dCas9-tracrRNA
- Used lab stock of T4 Ligase and buffer rather than iGEM stock
- 300 ng pSB1C3 = 3 uL
- 100 ng dCas9-tracrRNA PCR = 1 uL
- 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
- BO and IO controls for each time
- Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)
Objective: Test GFP repression by crRNAs
Transform pSB6A1-K608012 into DH5alpha-ZI
- Plated on LB+Amp
Objective: Obtain constructs for various uses
Streaked plate from frozen stock
- One LB+Cm plate in thirds:
- pSB1C3-R0040-antitracrRNA
- pSB1C3-Repeat-seq scaffold
- pSB1C3-Csy4-gRNA scaffold
Objective: Measurement interlab study
Cultured colonies (2 each)
- pSB1C3-E0240
- pSB1C3-K823005
- pSB1C3-K823012
- pSB3K3-I20260
July 25
Objective: Create pSB1C3-R0011-I13500
Plate results:
- no apparent difference between 15, 30, 45, or 60 minute ligations
- many colonies on all Experimental and BO control plates
- no colonies on IO control
Colony PCR:
- 16 colonies from pSB1C3-R0011-I13500 plates
- 4 from each ligation time
- Taq polymerase with oligos SB1C3-up and SB1C3-dn
- 66C anneal temp, 45 sec extension
Agarose gel of colony PCR
- No successes
- It seems as though colony PCRs are not working--try control with a gradient
Gradient Colony PCR
- to optimize with SB1C3-up and SB1C3-dn primers
- template colonies of pSB1C3-K608011 from plate
- anneal temps 62-72 C, extension time 45 sec
Agarose gel of gradient colony PCR
- left to right: 62C to 72C anneal temps
- Results: 62C was the best anneal temp--worked well
- gradual decrease as temp increased, no amplification above 65C
Objective: Switch dCas9-tracrRNA into pSB1C3
Plate results:
- no apparent difference between 15, 30, 45, or 60 minute ligations
- many colonies on all Experimental and BO control plates
- no colonies on IO control
Colony PCR:
- 16 colonies from pSB1C3-dCas9-tracrRNA plates
- 4 from each ligation time
- Taq polymerase with oligos SB1C3-up and SB1C3-dn
- 66C anneal temp, 45 sec extension
Agarose gel of colony PCR
- No successes
- It seems as though colony PCRs are not working--try control with a gradient
- see gradient results above
Objective: Various ligations
Prep-scale digests
- pSB1C3-Csy4-scaffold (2 copies) with XbaI/PstI
- to ligate into R0040
- pSB1C3-Repeat-seq scaffold (2 copies) with XbaI/PstI
- to ligate into R0040
- pSB1C3-R0040-anti-tracrRNA (2 copies) with XbaI/PstI
- 100 uL reactions each tube
PCR cleanups on all digests
- Qiagen kit
- Concentrations (in 50 uL each):
- pSB1C3-Csy4-scaffold: 55.9, 105.5 ng/uL
- pSB1C3-Repeat-seq scaffold: 15.2, 55.2 ng/uL
- pSB1C3-R0040-anti-tracrRNA: 90.6 ng/uL
- Need to extract from gels before using, only cleaned up for safer storage
Objective: Measurement interlab study
Minipreps
- Qiagen kit
- pSB1C3-K823005 (2 copies)
- concentrations 256.3, 314.8 ng/uL
- pSB1C3-K823012 (2 copies)
- concentrations 204.4, 192.5 ng/uL
- pSB1C3-E0240 (2 copies)
- concentrations 266.3, 241.5 ng/uL
Prep-scale digests
- pSB1C3-K823005 (2 copies) with SpeI/PstI
- pSB1C3-K823012 (2 copies) with SpeI/PstI
- pSB1C3-E0240 (2 copies) with XbaI/PstI
- 100 uL each tube
PCR cleanups on all digests
- Qiagen kit
- Concentrations (in 50 uL each):
- pSB1C3-K823005: 156.3, 181.7 ng/uL
- pSB1C3-K823012: 110.0, 98.0 ng/uL
- pSB1C3-E0240: 110.0, 114.6 ng/uL
Objective: Test crRNA repression of GFP reporter
Cultured colony
- To make CCEC of reporter strain
- One colony of pSB6A1-K608012 in DH5alpha-ZI
- Grew for 8 hrs during the day in 2 mL LB+Amp at 37C
- Inoculated into 100 mL flask of LB+Amp for overnight shaker at rt
Flow cytometry of reporter strain
- quick check to make sure reporter still works with new backbone
- Used parameters from previous GFP flow data
- Levels slightly under what was seen before, but still strong