General techniques: plasmid/PCR purification, inoculation, gel extraction, restriction & ligation.
Safety and waste disposal training
Introduction to CloneManager
General preparations: sterile mQ, sterile eppendorf
August
Week 1
Experiment 1
Inoculate E.Coli DH5a + pTGD-ccdA-Pmb1GFP-CmFRT
Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- 362.4 ng/µL
Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp
Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL
PCR on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589 ->HiFi PCR using Primestar polymerase. Analytical gel: negative ->PCR using Q5 polymerase. Analytical gel: negative ->PCR using Roche. Analytical gel: negative ->Touchdown PCR using Q5 polymerase. Analytical gel: negative
Control plasmid pTGD-ccdA-PMbAFGP-CmFRT: RD with AclI. Expected fragments: 758 bp, 1730 bp and 3956 bp. Analytical gel: negative --> Problem with plasmid pTGD-ccdA-PMbAFGP-CmFRT
Transformation: Knock in with lineair back-up DNA by electroporation. Incubation of the transformed cells and transfer the culture on a Cm plate.
Colony PCR on 48 colonies using crimson taq polymerase with two primer pairs: MDM0141/MDM0010 and MDM0046/MDM123. Expected fragments: 550bp & 2450 bp. Analytic gel: 4 positives
Colony PCR on 4 positive and 4 negative colonies using crimson taq polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
2 Colony PCR on 4 positive and 4 negative colonies using Taq and Phire polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
Colony PCR on 4 positive and 4 negative colonies using Emerald polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: 1 positive: colonie 35 .
Experiment 2
Inoculate E. coli DH5a + p5SpFRT-T7ccdB Inoculate E. coli DH5a + p10SpFRT-T7ccdB Inoculate E. coli DH5a + p20SpFRT-T7ccdB
Purify plasmids using Qiagen spin mini kit: nanodrop p5SpFRT-T7ccdB: 119,6 ng/µl p10SpFRT-T7ccdB: 156,3 ng/µl p20SpFRT-T7ccdB: 392,9 ng/µl
CcdB operon:
-> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB
operon
-> Purification of CcdB operon PCR fragment using Qiagen Qiaquick PCR purification kit and checked on analytical gel
Vectors pSB4A5,pSB3T5 and pSB6A1:
-> Resuspend plasmids from the iGEM kit
-> Transform in E.Coli Top10 subcloning cells using heat shock
-> Plate pSB4A5 and pSB6A1 on ampicillin plate and pSB3T5 on tetracyclin
and grow overnight at 37°C
Inoculation colonies of pSB3T5 and pSB6A1
Vector pSB4A5 and pSB6A1:
-> resuspend plasmid pSB4A5 and pSB6A1 from the iGEM kit
-> transform in E. coli Top10 subcloning cells (heat shock)
-> plate transformation on ampicillin plate and grow overnight at 37°C
inoculate pSB3T5 and pSB6A1 again and in warm chamber (37°C).
Experiment 6
CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
nanodrop: 301,5 ng/µl
pSB1C3 -> Inoculate E. coli DH5a + pSB1C3
-> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl
Restriction of CcdB operon and pSB1C3 with XbaI and PstI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 16.9 ng/µl
RD_pSB1C3: 12.3 ng/µl
Ligation of CcdB operon and pSB1C3
Transformation in E.Coli Top10 subcloning cells using heat shock
and incubation on chloramphenicol agar plates at 37°C: negative
Week 2
Experiment 1
Experiment 2
Experiment 6
Again transformation in E.Coli Top10 subcloning cells using heat shock
and incubation on chloramphenicol agar plates: negative
CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
Again restriction of CcdB operon and pSB1C3 with XbaI and PstI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 16.6 ng/µl
RD_pSB1C3: 17.7 ng/µl
Ligation of CcdB operon and pSB1C3: overnight at 16°C
Transformation in E. coli DH5a using heat shock
and incubation on chloramphenicol agar plates + glucose at 30°C: negative
Restriction of CcdB operon and pSB1C3 with XbaI and PstI, using DpnI
Control of restriction fragments: positive, so we hope that ligation will succeed
Ligation of CcdB operon and pSB1C3: overnight at 16°C
Transformation in E. coli DH5a
and incubation on chloramphenicol agar plates: negative
Week 3
Experiment 1
Experiment 2
Experiment 6
Again transformation in E. coli DH5a (because we are shore that the fragments were present for ligation and so will be succeed)
and incubation on chloramphenicol agar plates: negative
Control of ligations (with PCR using primers MDM0606 and MDM0607): negative
Ligation of CcdB operon and pSB1C3: 15 minutes at 16°C
Again transformation in E. coli DH5a
and incubation on chloramphenicol agar plates: negative
CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
Again restriction of CcdB operon and pSB1C3 with XbaI and PstI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 15.3 ng/µl
RD_pSB1C3: 16.8 ng/µl
Again ligation of CcdB operon and pSB1C3: 1 hour at room temperature
Transformation in E. coli DH5a
and incubation on chloramphenicol agar plates: positive
Colony PCR of colonies: negative
Designing primers for Gibson Assembly
Week 4
Experiment 2
Q5 PCR of CcdB operon and plasmids (pSB3T5 and pSB4A5), using the designed Gibson primers
Purification of PCR-product after using DpnI
Control of fragments on a gel => Backbones are present, CcdB operons not
PrimeStar PCR of CcdB operons, using the designed Gibson primers
Purification of PCR-product after using DpnI
Control of fragments on a gel => CcdB operons are still not present
Q5 PCR of CcdB operons on a linear CcdB operon, using the designed Gibson primers
Purification of PCR-product
Control of fragments on a gel => CcdB operon is visible
Gibson Assembly (1h at 50°C)
Transformation in E. coli DH5a using heat shock (42°C)
and plate pSB4A5 on an ampicillin plate and pSB3T5 on tetracyclin plate at 37°C: positive Strange phenomena: most colonies are red, let’s control it
Colony PCR of colonies (derived from Gibson Assembly): negative
Experiment 3
Experiment 6
While waiting for the Gibson Assembly primers
Again restriction of CcdB operon and pSB1C3 using XbaI and PstI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 10.5 ng/µl
RD_pSB1C3: 7.2 ng/µl
Ligation of CcdB operon and pSB1C3: overnight at 16°C
Transformation in E. coli DH5a using heat shock
and incubation on chloramphenicol agar plates + glucose at 30°C: negative
After receiving the primers for Gibson Assembly
Q5 PCR of CcdB operon and pSB1C3, using the designed Gibson primers Purification of PCR-product after using DpnI
Control of fragments on a gel => Backbone is present, CcdB operon not
PrimeStar PCR of CcdB operon, using the designed Gibson primers
Purification of PCR-product after using DpnI
Control of fragments on a gel => CcdB operon is not present
Q5 PCR of CcdB operon on a linear CcdB operon, using the designed Gibson primers
Purification of PCR-product
Control of fragments on a gel => CcdB operon is visible
Gibson Assembly (1h at 50°C)
Transformation in E. coli DH5a using heat shock (42°C)
and incubation on chloramphenicol agar plates at 37°C: positive Strange phenomena: colonies are red, let’s control it
Colony PCR of colonies (derived from Gibson Assembly): negative
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