Team:UGent/Labjournal

From 2013.igem.org

(Difference between revisions)
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<li> Experiment 1 </li>
<li> Experiment 1 </li>
<ul class="a">
<ul class="a">
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<li> Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- 362.4 ng/µL </li>
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<li> Inoculate <i> E.Coli </i>DH5a + pTGD-ccdA-Pmb1GFP-CmFRT </li>
 +
<li> Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- <b>362.4 ng/µL</b> </li>
<li> Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp </li>
<li> Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp </li>
<li> Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL</li>
<li> Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL</li>

Revision as of 17:11, 27 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
    • 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
    • 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
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