Team:Caltech/Week 5
From 2011.igem.org
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__NOTOC__ | __NOTOC__ | ||
==July 11== | ==July 11== | ||
+ | [[File:7-11gelpNT003assem.jpg|thumb|left|1 ladder, 2 R0010, 3 K123003, 4 B0014, 5 pSB4A5]] | ||
<p>Miniprep pSB4A5<br/> | <p>Miniprep pSB4A5<br/> | ||
Run PCR to amplify [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123003 K123003], [http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_PSB4A5 PSB4A5]<br/> | Run PCR to amplify [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123003 K123003], [http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_PSB4A5 PSB4A5]<br/> | ||
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===Results=== | ===Results=== | ||
The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. | The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. | ||
- | |||
==July 12== | ==July 12== | ||
+ | [[File:7-12pcrgel.jpg|thumb|from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001]] | ||
Transformed competent cells with pSB3C5. | Transformed competent cells with pSB3C5. | ||
Ran PCR of parts for pNT001 and pNT002 Gibson assembly. | Ran PCR of parts for pNT001 and pNT002 Gibson assembly. | ||
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===Results=== | ===Results=== | ||
All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified. | All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified. | ||
- | [[File:7- | + | |
+ | ==July 13== | ||
+ | Retrieved and imaged pulsed field gel.<br> | ||
+ | Ran PCR of R0040 and B0014 with primers that failed yesterday.<br> | ||
+ | Purified PCR products for Gibson of pNT001 and pNT003.<br> | ||
+ | Started overnights of pSB3C5.<br> | ||
+ | Replated some pSB3C5<br> | ||
+ | ===Results=== | ||
+ | <p>The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.</p> | ||
+ | The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plateand starting overnights to send off for sequencing to make sure the plasmid is okay.<br> | ||
+ | We redid the PCR of R0040 and B0014, but no bands appeared in the gel.<br> | ||
+ | [[File:7-13pulsedgel.jpg|thumb|lanes 1 lambda mono cut ladder; 2 blank; 3 fosmid kit control; 4 LA River location 9; LA River location 10]] | ||
+ | PCR Purifications for Gibson | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Part</th> | ||
+ | <th>Concentration (ng/ul)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R0010 for pNT003</td> | ||
+ | <td>130.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123003</td> | ||
+ | <td>99.9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB4A5 + prefix and suffix</td> | ||
+ | <td>90.4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R0010 for pNT001</td> | ||
+ | <td>142.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123000</td> | ||
+ | <td>111.4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123001</td> | ||
+ | <td>123.0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>B0014 for pNT003</td> | ||
+ | <td>119.9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>B0014 for pNT001</td> | ||
+ | <td>139.7</td> | ||
+ | </tr> | ||
+ | </table> | ||
}} | }} |
Revision as of 07:29, 14 July 2011
Project |
July 11Miniprep pSB4A5 ResultsThe PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. July 12Transformed competent cells with pSB3C5. Ran PCR of parts for pNT001 and pNT002 Gibson assembly. Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2. ResultsAll of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified. July 13Retrieved and imaged pulsed field gel. ResultsThe pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters. The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plateand starting overnights to send off for sequencing to make sure the plasmid is okay. PCR Purifications for Gibson
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