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Revision as of 10:28, 26 August 2013

Journal

July

Week 3

• Introduction given by our lab instructors
• 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 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
• 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
• cPCR of colonies: negative
• Designing primers for Gibson Assembly

Days: Hours: Mins: Secs: Until Jamboree Lyon Tweets van @iGEM_UGent