Team:UGent/Labjournal
From 2013.igem.org
(Difference between revisions)
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</ul> | </ul> | ||
<BR> | <BR> | ||
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<li> Experiment 2 </li> | <li> Experiment 2 </li> | ||
<ul class="a"> | <ul class="a"> | ||
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<BR> -> Resuspend plasmids from the iGEM kit | <BR> -> Resuspend plasmids from the iGEM kit | ||
<br> -> Transform pSB1C3 in <i>E. coli</i> Top10 subcloning cells, using heat shock (42°C) | <br> -> Transform pSB1C3 in <i>E. coli</i> Top10 subcloning cells, using heat shock (42°C) | ||
- | <br>-> Plate on chloramphenicol plate and grow overnight at 37°C | + | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C |
<br> -> Inoculate <i>E. coli</i> Top10 + pSB1C3 | <br> -> Inoculate <i>E. coli</i> Top10 + pSB1C3 | ||
<br> -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl</li> | <br> -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl</li> | ||
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<li> Experiment 6 </li> | <li> Experiment 6 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> | + | <li> New <b>transformation</b> in <i> E. coli </i> Top10 subcloning cells, using heat shock (42°C) |
<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | ||
<li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon</li> | <li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon</li> | ||
- | <li> | + | <li> New <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI |
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br> Restriction Digest of T7-<i>ccdB</i>: 16.6 ng/µl | <br> Restriction Digest of T7-<i>ccdB</i>: 16.6 ng/µl | ||
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<li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) | <li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) | ||
<br> -> Plate on chloramphenicol + glucose plate and grow overnight at 30°C: <b>negative</b> | <br> -> Plate on chloramphenicol + glucose plate and grow overnight at 30°C: <b>negative</b> | ||
- | <li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | + | <li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI |
- | <br> -> Purification by using minElute reaction cleanup kit | + | <br> Because a preparative gel purification cause low concentrations, we will use DpnI |
+ | <br> -> Purification by using minElute reaction cleanup kit</li> | ||
<li> Control of restriction fragments: <b>positive</b>, so we hope that ligation will succeed</li> | <li> Control of restriction fragments: <b>positive</b>, so we hope that ligation will succeed</li> | ||
<li> <b>Ligation</b> of CcdB operon and pSB1C3: overnight at 16°C</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, using heat shock (42°C) | <li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) | ||
<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | ||
+ | <br> (it does not work, maby our transformation is not succeed) | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<h3> Week 3 </h3> | <h3> Week 3 </h3> | ||
<ul> | <ul> | ||
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<li> Experiment 6 </li> | <li> Experiment 6 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> | + | <li> New <b>transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) |
- | <br> (because we are sure that the fragments were present for ligation and so will | + | <br> (because we are sure that the fragments were present for ligation and so will lead to succesful results) |
- | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> </li> | + | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> (it still does not work) </li> |
<li> Control of ligations by using PCR with primers MDM0606 and MDM0607: <b>negative</b> </li> | <li> Control of ligations by using PCR with primers MDM0606 and MDM0607: <b>negative</b> </li> | ||
- | <li> <b>Ligation</b> of CcdB operon and pSB1C3: 15 minutes at 16°C</li> | + | <li> <b>Ligation</b> again of CcdB operon and pSB1C3: 15 minutes at 16°C</li> |
- | <li> | + | <li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) |
<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> </li> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> </li> | ||
- | <li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon</li> | + | <li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon |
- | <li> | + | <br> because we have no more T7<i>ccdB</i> fragments</li> |
+ | <li> New <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | ||
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br> Restriction Digest of T7-<i>ccdB</i>: 15.3 ng/µl | <br> Restriction Digest of T7-<i>ccdB</i>: 15.3 ng/µl | ||
<br> Restriction Digest of pSB1C3: 16.8 ng/µl</li> | <br> Restriction Digest of pSB1C3: 16.8 ng/µl</li> | ||
- | <li> | + | <li> <b>Ligation</b> of CcdB operon and pSB1C3: 1 hour at room temperature</li> |
<li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) | <li> <b>Transformation</b> in <i>E. coli</i> DH5a, using heat shock (42°C) | ||
- | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> </li> | + | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> (finally)</li> |
- | <li> <b>Colony PCR</b> (Taq): <b>negative</b> | + | <li> <b>Colony PCR</b> (Taq): <b>negative</b> |
- | <li> Designing primers for Gibson Assembly </li> | + | <br> It seems that restriction-ligation does not work, thus we will use another technique, called Gibson Assembly </li> |
+ | <li> Designing primers for Gibson Assembly for the insert T7<i>ccdB</i> and the backbone pSB1C3 </li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<ul class="a"> | <ul class="a"> | ||
<b>=> While waiting for the Gibson Assembly primers </b> | <b>=> While waiting for the Gibson Assembly primers </b> | ||
- | <li> Again <b>restriction</b> of CcdB operon and pSB1C3 | + | <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> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br> Restriction Digest of T7-<i>ccdB</i>: 10.5 ng/µl | <br> Restriction Digest of T7-<i>ccdB</i>: 10.5 ng/µl | ||
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<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> </li> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> </li> | ||
<li> <b>Colony PCR</b> (Taq) of these colonies: <b>negative</b> | <li> <b>Colony PCR</b> (Taq) of these colonies: <b>negative</b> | ||
- | <br> <br> </li> | + | <br> |
+ | <br> </li> | ||
<b>=>After receiving the primers for Gibson Assembly</b> | <b>=>After receiving the primers for Gibson Assembly</b> | ||
<li> <b>Q5 PCR</b> of CcdB operon and pSB1C3, using the designed Gibson primers | <li> <b>Q5 PCR</b> of CcdB operon and pSB1C3, using the designed Gibson primers | ||
- | <br> -> Purification of PCR-product after DpnI treatment by using minElute reaction cleanup kit | + | <br> -> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit |
<br> -> Control of fragments on a gel => Backbone is present, CcdB operon not </li> | <br> -> Control of fragments on a gel => Backbone is present, CcdB operon not </li> | ||
<li> <b>PrimeStar PCR</b> of CcdB operon, using the designed Gibson primers | <li> <b>PrimeStar PCR</b> of CcdB operon, using the designed Gibson primers | ||
- | <br> -> Purification of PCR-product after DpnI treatment by using minElute reaction cleanup kit | + | <br> -> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit |
<br> -> Control of fragments on a gel => CcdB operon is not present </li> | <br> -> Control of fragments on a gel => CcdB operon is not present </li> | ||
<li> <b>Q5 PCR</b> of CcdB operon on a linear CcdB operon, using the designed Gibson primers | <li> <b>Q5 PCR</b> of CcdB operon on a linear CcdB operon, using the designed Gibson primers | ||
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<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> | ||
<br> Strange phenomena: colonies are red, let’s control it </li> | <br> Strange phenomena: colonies are red, let’s control it </li> | ||
- | <li> <b>Colony PCR</b> (Taq) of colonies derived from Gibson Assembly: <b>negative</b> </li> | + | <li> <b>Colony PCR</b> (Taq) of colonies derived from Gibson Assembly: <b>negative</b> |
+ | <br> We think that some original plasmid has to be present | ||
+ | <br> and the bacteria love that plasmid more than the plasmid with <i>ccdB</i></li> | ||
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<li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the linearised plasmid | <li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the linearised plasmid | ||
<br> -> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: nanodrop: | <br> -> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: nanodrop: | ||
- | <br>pSB1C3-backbone: 109.9 ng/µl</li> | + | <br> pSB1C3-backbone: 109.9 ng/µl</li> |
<li> <b>Gibson Assembly</b> (1h at 50°C)</li> | <li> <b>Gibson Assembly</b> (1h at 50°C)</li> | ||
<li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.4) | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.4) | ||
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<li>Plasmid purification of the 6 colonies + restriction to control of they are the good colonies (on a gel) | <li>Plasmid purification of the 6 colonies + restriction to control of they are the good colonies (on a gel) | ||
<br> There is a possibility that they are the good ones, so we will send it to sequenate | <br> There is a possibility that they are the good ones, so we will send it to sequenate | ||
- | <br> -> pSB1C3-T7<i>ccdB</i> sent for sequencing with primers MDM0606, MDM0607, MDM0060 and MDM0039: <b>negative</b> </li> | + | <br> -> pSB1C3-T7<i>ccdB</i> sent for <b>sequencing</b> with primers MDM0606, MDM0607, MDM0060 and MDM0039: <b>negative</b> </li> |
<li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the linearised plasmid | <li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the linearised plasmid | ||
<br>-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: | <br>-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: |
Revision as of 19:39, 6 September 2013
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