Team:UGent/Labjournal

From 2013.igem.org

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{{:Team:UGent/Templates/Header}}
{{:Team:UGent/Templates/Header}}
<html>
<html>
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<h1> July </h1>
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<h1> Journal</h1>
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<h2> Week 3 </h2>
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<h2> July </h2>
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<h3> Week 3 </h3>
<ul>
<ul>
<li> Introduction given by our lab instructors</li>  
<li> Introduction given by our lab instructors</li>  
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</ul>
</ul>
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<h1> August </h1>
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<h2> August </h2>
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<h2> Week 1 </h2>
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<h3> Week 1 </h3>
<ul>
<ul>
<li> Experiment 1 </li>
<li> Experiment 1 </li>
<ul class="a">
<ul class="a">
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<li> <b>Purify plasmid</b> pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- <b>362.4 ng/µL</b> </li>
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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> <b>Preparative Restriction Digest (RD)</b> of purified plasmid: BspHI & BstAPI: expected fragments of <b>5541bp and 903 bp </b></li>
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<li> Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp </li>
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<li> <b>Gel purify RD-fragment of 5541 bp </b> using Qiagen Qiaquick gel extraction kit: Nanodrop -- <b>70.4 ng/µL</b></li>
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<li> Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL</li>
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<li> HiFi <B>PCR using Primestar polymerase</B> on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b>negative </b></li>
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<li> HiFi PCR using Primestar polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b>negative </b></li>
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<li> <b>PCR using Q5 polymerase </b> on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
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<li> PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
<li> PCR using Roche on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
<li> PCR using Roche on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
<li> Touchdown PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
<li> Touchdown PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT  with MDM588 & MDM589. Analytical gel: <b> negative </b></li>
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<h2> Week 2 </h2>
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<h3> Week 2 </h3>

Revision as of 16:21, 18 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

  • Experiment 6

    • Week 2

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