IGEM:MIT/2006/Notebook/2006-8-10: Difference between revisions
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#PCR pchBA with 5% Betane -'''done''' | #PCR pchBA with 5% Betane -'''done''' | ||
#site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -'''done''' | #site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -'''done''' | ||
==Indole KO Strain== | ==Indole KO Strain== | ||
Revision as of 14:26, 10 August 2006
to do
- GC work with new smell samples -rescheduled for 8/11
- Miniprep all LCs/make appropriate glycerols/sequence -done
- Remember to Make 10 biobrick named glycerols and send for sequencing -done
- Make LCs of IK transformants in pm (overnight smell test and regular batch for glycerols/am smell test) -done
- PCR pchBA with 5% Betane -done
- site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -done
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
