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
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
HiFi PCR using Primestar polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
PCR using Roche on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
Touchdown PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
Control plasmid pTGD-ccdA-PMbAFGP-CmFRT: RD with AclI. Expected fragments:.............. Analytical gel: negative --> Problem with plasmid pTGD-ccdA-PMbAFGP-CmFRT
Transformation: Knock in with lineair 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 plasmid pTGD-ccdA-Pmb1GFP-CmFRT 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
Vector pSB4A5:
-> Resuspend plasmid pSB4A5 from the iGEM kit (plate 5, well1)
-> Transorm in E.Coli Top10 subcloning cells using heat shock
-> Plate on ampicillin plate and grow overnight at 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 DH5a
and incubation on chloramphenicol agar plates at 37°C: negative
Week 2
Experiment 1
Experiment 2
Experiment 6
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: 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
and incubation on chloramphenicol agar plates + glucose at 30°C: negative
Control of restriction fragments: positive
Restriction of CcdB operon and pSB1C3 with XbaI and PstI, using DpnI
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
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
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