Team:Caltech/Week 7
From 2011.igem.org
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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/> | 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/> | ||
+ | Plated enrichment cultures on LB<br/> | ||
===Results=== | ===Results=== | ||
<table border="1"> | <table border="1"> | ||
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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/> | 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/> | PCR pSB3K3 to linearizer it for Gibson assembly<br/> | ||
- | + | Continued attempts to spread chemical solution on minimal media plates<br/> | |
Minimal media transfers <br/> | Minimal media transfers <br/> | ||
===Results=== | ===Results=== |
Revision as of 18:46, 28 July 2011
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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 ResultsThe 16s PCR from yesterday, shown in the gel today, had a band in the first lane of negative controls, indicating contamination.
July 28Plate packaged fosmids to obtain titer
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