From 2013.igem.org

Revision as of 14:31, 14 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 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

Week 2

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