Team:UGent/Labjournal
From 2013.igem.org
(Difference between revisions)
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<br> Strange phenomena: most colonies are red, let’s control it </li> | <br> Strange phenomena: most colonies are red, let’s control it </li> | ||
<li> <b>Colony PCR</b> of colonies (derived from Gibson Assembly): <b>negative</b> </li> | <li> <b>Colony PCR</b> of colonies (derived from Gibson Assembly): <b>negative</b> </li> | ||
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</ul> | </ul> | ||
<br> | <br> | ||
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<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 using DpnI | + | <br> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit, after using DpnI |
<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 | ||
- | <br> Purification of PCR-product | + | <br> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit |
<br> Control of fragments on a gel => CcdB operon is visible </li> | <br> Control of fragments on a gel => CcdB operon is visible </li> | ||
<li> <b>Gibson Assembly</b> (1h at 50°C)</li> | <li> <b>Gibson Assembly</b> (1h at 50°C)</li> | ||
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<br>Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br>Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br>RD_T7-ccdB: 14.4 ng/µl | <br>RD_T7-ccdB: 14.4 ng/µl | ||
- | <br>RD_pSB1C3: 4.4 ng/µl | + | <br>RD_pSB1C3 : 4.4 ng/µl |
<li> <b>Ligation</b> of CcdB operon and pSB1C3: 25 minutes at room temperature </li> | <li> <b>Ligation</b> of CcdB operon and pSB1C3: 25 minutes at room temperature </li> | ||
<li> <b>Transformation</b> in E. coli DH5a using heat shock | <li> <b>Transformation</b> in E. coli DH5a using heat shock | ||
<br>and incubation on chloramphenicol agar plates at 37°C: <b>negative</b> </li> | <br>and incubation on chloramphenicol agar plates at 37°C: <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: nanodrop: pSB1C3-backbone: 109.9 ng/µl</li> | + | <br>Purification of PCR-product: nanodrop: |
+ | <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 E. coli DH5a using electroporation (Time Constant: 4.4) | <li> <b>Transformation</b> in E. coli DH5a using electroporation (Time Constant: 4.4) | ||
<br>and incubation on chloramphenicol agar plates at 37°C: <b>positive</b> </li> | <br>and incubation on chloramphenicol agar plates at 37°C: <b>positive</b> </li> | ||
- | <li> <b>Colony PCR</b> (Crimson) of colonies: 6 colonies can possible be <b>positive</b> (but it is not sure)</li> | + | <li> <b>Colony PCR</b> (Crimson) of colonies: |
+ | <br>6 colonies can possible be <b>positive</b> (but it is not sure)</li> | ||
<li>Control of fragment of pSB1C3 on a gel => Backbone is not present</li> | <li>Control of fragment of pSB1C3 on a gel => Backbone is not present</li> | ||
<li>Inoculate the 6 colonies (overnight)</li> | <li>Inoculate the 6 colonies (overnight)</li> | ||
- | <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 ons, so we send it to sequenate | + | <br> There is a possibility that they are the good ons, so we send it to sequenate </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 | ||
- | Purification of PCR-product: nanodrop: pSB1C3-backbone: 58.0 ng/µl | + | <br>Purification of PCR-product using Qiagen Qiaquick PCR purification kit: |
+ | <br> nanodrop: pSB1C3-backbone: 58.0 ng/µl | ||
<br> Control of fragment of pSB1C3 on a gel => Backbone is not present</li> | <br> Control of fragment of pSB1C3 on a gel => Backbone is not present</li> | ||
<li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the restriction digest of pSB1C3 | <li> <b>Q5 PCR</b> of pSB1C3, using the designed Gibson primers and the restriction digest of pSB1C3 | ||
- | <br>Purification of PCR-product: nanodrop: pSB1C3-backbone: 76.2 ng/µl | + | <br>Purification of PCR-product using Qiagen Qiaquick PCR purification kit: |
+ | <br>nanodrop: pSB1C3-backbone: 76.2 ng/µl | ||
<br> Control of fragment of pSB1C3 on a gel => Backbone is present</li> | <br> Control of fragment of pSB1C3 on a gel => Backbone is present</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 E. coli DH5a using electroporation (Time Constant: 4.3) </li> | <li> <b>Transformation</b> in E. coli DH5a using electroporation (Time Constant: 4.3) </li> | ||
and incubation on chloramphenicol agar plates at 37°C: <b>positive</b> => many colonies</li> | and incubation on chloramphenicol agar plates at 37°C: <b>positive</b> => many colonies</li> | ||
- | <li> <b>Colony PCR</b> (Crimson) of colonies: </li> | + | <li> <b>Colony PCR</b> (Crimson) of colonies: |
+ | <br>we tested 48 colonies and 4 colonies show the right fragment </li> | ||
+ | <li> Inoculate the 4 good colonies</li> | ||
+ | <li>Plasmid purification of the 4 colonies + restriction to control of they are the good colonies (on a gel) </li> | ||
Revision as of 18:26, 5 September 2013
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