IGEM:MIT/2006/Notebook/2006-8-10: Difference between revisions
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#Regrew THI3 culture because not enough cells | #Regrew THI3 culture because not enough cells | ||
#Mutagenesis on 4 ATF1 (200,220,250,270) | #Mutagenesis on 4 ATF1 (200,220,250,270) | ||
#Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis | |||
Revision as of 09:06, 10 August 2006
to do
- GC work with new smell samples
- Miniprep all LCs/glycerols
- Make 10 biobrick named glycerols and send for sequencing
- Make LCs of IK transformants
- plate reader test
- PCR pchBA with 5% Betane
- site-directed mutagenesis on banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers
- clone WGD into pseudomonas shuttle vector
Indole KO Strain
The following table summarizes the results of the indole KO transformations.
| # of Transformants | Notes about colonies | |
|---|---|---|
| pUC18 | lots | essentially uniform in size (though denser areas contain smaller colonies); well spread across plate |
| J45100 | ~110 | many tended to grow in clusters; range of sizes from very small to large (though large colonies may be more than one) |
| J45120 | ~70 | not as many clusters; the colonies are huge, in general (a few small ones, though) |
| J45170 | ~100 | not as many clusters; the colonies are huge, in general (a few small ones, though) |
| J45200 | ~110 | range of sizes from very small to large; some clusters |
| J45220 | ~50 | a few small colonies but most are huge; few clusters |
Mutagenesis
- Held off on BAT2 because of Spe1 site
- Regrew THI3 culture because not enough cells
- Mutagenesis on 4 ATF1 (200,220,250,270)
- Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis
