Team:UGent/Labjournal

From 2013.igem.org

(Difference between revisions)
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<li> Experiment 6 </li>
<li> Experiment 6 </li>
<ul class="a">
<ul class="a">
-
<li> Transform the selected plasmids from the distribution DNA kit in E. coli DH5a subcloning cells</li>
+
<li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
-
<li> Inoculate E. coli DH5a + pSB1C3</li>
+
<br> nanodrop: 301,5 ng/µl </li>
-
<li> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 301,5 ng/µl</li>
+
<li> <b>pSB1C3</b>
 +
<br> -> Inoculate <i>E. coli</i> DH5a + pSB1C3
 +
<br> -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl</li>
 +
<li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI<br>
 +
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
 +
<br> RD_T7-ccdB: 16.9 ng/µl
 +
<br> RD_pSB1C3: 12.3 ng/µl</li>
 +
<li> <b>Ligation</b> of CcdB operon and pSB1C3</li>
 +
<li> <b>Transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates at 37°C: <b>negative</b>
</ul>
</ul>
-
 
+
</ul>
 +
<br>
<h3> Week 2 </h3>
<h3> Week 2 </h3>
 +
<ul>
 +
<li> Experiment 1 </li>
 +
<ul class="a">
 +
</ul>
 +
<br>
 +
<li> Experiment 2 </li>
 +
<ul class="a">
 +
</ul>
 +
<br>
 +
<li> Experiment 6 </li>
 +
<ul class="a">
 +
<li> Again <b>transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates: <b>negative</b>
 +
<li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon</li>
 +
<li> Again <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI<br>
 +
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
 +
<br> RD_T7-ccdB: 16.6 ng/µl
 +
<br> RD_pSB1C3: 17.7 ng/µl</li>
 +
<li> <b>Ligation</b> of CcdB operon and pSB1C3: overnight at 16°C</li>
 +
<li> <b>Transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates + glucose at 30°C: <b>negative</b>
 +
<li> Control of restriction fragments: <b>positive</b></li>
 +
<li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI, using DpnI</li>
 +
<li> <b>Ligation</b> of CcdB operon and pSB1C3: overnight at 16°C</li>
 +
<li> <b>Transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates: <b>negative</b>
 +
</ul>
 +
</ul>
 +
<h3> Week 3 </h3>
 +
<ul>
 +
<li> Experiment 1 </li>
 +
<ul class="a">
 +
</ul>
 +
<br>
 +
<li> Experiment 2 </li>
 +
<ul class="a">
 +
</ul>
 +
<br>
 +
<li> Experiment 6 </li>
 +
<ul class="a">
 +
<li> Again <b>transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates: <b>negative</b> </li>
 +
<li> Control of ligations (with PCR using primers MDM0606 and MDM0607): <b>negative</b> </li>
 +
<li> <b>Ligation</b> of CcdB operon and pSB1C3: 15 minutes at 16°C</li>
 +
<li> Again <b>transformation</b> in <i>E. coli</i> DH5a
 +
<br> and incubation on chloramphenicol agar plates: <b>negative</b> </li>
 +
 +
</ul>
 +
</ul>
</html>
</html>
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Revision as of 09:41, 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 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
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