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Team:Caltech/Week 7
From 2011.igem.org
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===Results=== | ===Results=== | ||
Decided not to use the pNT003 PCR'd insert, as the band of the correct length was much fainter than a lower length band. Chose not to do Gibson assembly of the positive GFP control, despite not having any green colonies last week, instead focusing on assembling pNT002 and pNT003.<br/> | Decided not to use the pNT003 PCR'd insert, as the band of the correct length was much fainter than a lower length band. Chose not to do Gibson assembly of the positive GFP control, despite not having any green colonies last week, instead focusing on assembling pNT002 and pNT003.<br/> | ||
| - | + | <gallery> | |
| - | + | File:7-25pcrgel1.jpg|lane 1 NEB 2-log ladder, 2-4 pSB4A5, 5-7 R0040, 8-10 K123001, 11-13 B0014 | |
| + | File:7-25pcrgel2.jpg|lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5 R0010, 6 K123003, 7 B0014, 8 blank, 9-11 GFP insert, 12 GFP constitutive promoter, 13 GFP, 14 GFP terminator, 15 NEB 2-log ladder | ||
| + | </gallery> | ||
PCR concentrations | PCR concentrations | ||
<table border="1"> | <table border="1"> | ||
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==July 26== | ==July 26== | ||
Packaging of 9mix and (10mix + 10-2) ligations<br/> | Packaging of 9mix and (10mix + 10-2) ligations<br/> | ||
| - | |||
Attempted various methods of spreading chemical solution on minimal media plates<br/> | Attempted various methods of spreading chemical solution on minimal media plates<br/> | ||
16s PCR<br/> | 16s PCR<br/> | ||
| + | Ran 20 ul Gibson reaction of pNT003 and a negative control. PCR purified these using the Qiaquick kit. Ran an Spe1 digest on part of the reaction. If the plasmid had formed, a cut with Spe1 would create a band of around 5kb.<br/> | ||
| + | ===Results=== | ||
| + | <table border="1"> | ||
| + | <tr> | ||
| + | <th>Part</th> | ||
| + | <th>Concentration (ng/ul)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>pNT003 +</td> | ||
| + | <td>27.8</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>pNT003 -</td> | ||
| + | <td>23.4</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | </table> | ||
| + | The gel gave no bands in any lane, with or without the restriction digest. pNT003 + without the Spe1 digest had a faint smear, the other lanes had nothing. Chose to transform anyway<br/> | ||
==July 27== | ==July 27== | ||
Grew up cells for titering of packaged fosmids-Reached OD600 of 0.974<br/> | Grew up cells for titering of packaged fosmids-Reached OD600 of 0.974<br/> | ||
| - | |||
Redid 16s PCR<br/> | Redid 16s PCR<br/> | ||
| - | + | Attempted to PCR the inserts for pNT002 and pNT003 together from their component parts. Did a gel extraction to get the correct length product<br/> | |
| + | PCR pSB3K3 to linearizer it for Gibson assembly<br/> | ||
Julia did something<br/> | Julia did something<br/> | ||
Minimal media transfers <br/> | Minimal media transfers <br/> | ||
| + | ===Results=== | ||
| + | <gallery> | ||
| + | File:7-2716sgel.jpg|lane 1 NEB 2-log ladder, 2-3 negative controls, 4-6 LA River sample 9, 7-9 LA River sample 10, 11-12 pET53DEST | ||
| + | File:7-27psb3k3.jpg|lane 1 NEB 2-log ladder, 2-4 pSB3K3 | ||
| + | </gallery> | ||
| + | <p>The 16s PCR from yesterday, shown in the gel today, had a band in the first lane of negative controls, indicating contamination.</p> | ||
| + | <p>The pSB3K3 amplification failed. The bands indicated a much lower length than expected. After doing an alignment in Geneious, it appears our pSB3N5 primers we ordered for pSB3C5, which we are no longer using because of our competent cell strain, do not work with earlier versions of the 3 origin plasmids.</p> | ||
| + | <p>Both the negative control and experimental of PCR purified Gisbson assembly from yesterday of pNT003 had 0 colonies. The transformation with 1 ul pUC 19 had no colonies.</p> | ||
| + | <table border="1"> | ||
| + | <tr> | ||
| + | <th>Part</th> | ||
| + | <th>Concentration (ng/ul)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>pNT002 insert</td> | ||
| + | <td>28.3</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>pNT003 insert</td> | ||
| + | <td>9.5</td> | ||
| + | </tr> | ||
| + | </table> | ||
==July 28== | ==July 28== | ||
Plate packaged fosmids to obtain titer <br/> | Plate packaged fosmids to obtain titer <br/> | ||
Revision as of 03:51, 28 July 2011
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Project |
July 24Started overnight cultures of pSB3K3 July 25PCR'd parts for pNT002: R0040, K123001, B0014, pSB4A5 ResultsDecided not to use the pNT003 PCR'd insert, as the band of the correct length was much fainter than a lower length band. Chose not to do Gibson assembly of the positive GFP control, despite not having any green colonies last week, instead focusing on assembling pNT002 and pNT003.
PCR concentrations
July 26Packaging of 9mix and (10mix + 10-2) ligations Results
The gel gave no bands in any lane, with or without the restriction digest. pNT003 + without the Spe1 digest had a faint smear, the other lanes had nothing. Chose to transform anyway July 27Grew up cells for titering of packaged fosmids-Reached OD600 of 0.974 Results
The 16s PCR from yesterday, shown in the gel today, had a band in the first lane of negative controls, indicating contamination. The pSB3K3 amplification failed. The bands indicated a much lower length than expected. After doing an alignment in Geneious, it appears our pSB3N5 primers we ordered for pSB3C5, which we are no longer using because of our competent cell strain, do not work with earlier versions of the 3 origin plasmids. Both the negative control and experimental of PCR purified Gisbson assembly from yesterday of pNT003 had 0 colonies. The transformation with 1 ul pUC 19 had no colonies.
July 28Plate packaged fosmids to obtain titer
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