IGEM:MIT/2006/Notebook/2006-8-10

to do

 * 1) GC work with new smell samples -rescheduled for 8/11
 * 2) Miniprep all LCs/make appropriate glycerols/sequence -done
 * 3) *Remember to Make 10 biobrick named glycerols and send for sequencing -done
 * 4) Make LCs of IK transformants in pm (overnight smell test and regular batch for glycerols/am smell test) -done
 * 5) PCR pchBA with 5% Betane -done
 * 6) site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -done

PCR of pchBA
...it worked! The following showed bands on the gel and are currently in the fridge (pcr cleaned up):
 * pchBA pcr'ed w/ new forward and reverse primers, with 2&mu;L betaine added to pcr rxn [53.3 ng/&mu;L]
 * pchBA pcr'ed w/ new forward and reverse primers, with 4&mu;L betaine added to pcr rxn [9.9 ng/&mu;L]
 * pchBA pcr'ed w/ new forward and reverse primers, with a 96deg (instead of 94deg) denature step...we stopped the reaction on cycle 21 out of 30 b/c we needed the block for mutagenesis [17.8 ng/&mu;L]
 * pchA pcr'ed w/ new reverse primer (and the old forward one), with 2 &mu;L betaine added to pcr rxn [64.2 ng/&mu;L]

next steps: biobrick, do the pchA mutagenesis, hook it up to terminator, RBS, promoter!

Indole KO Strain
The following table summarizes the results of the indole KO transformations.

Mutagenesis

 * 1) Held off on BAT2 because of Spe1 site
 * 2) Regrew THI3 culture because not enough cells
 * 3) Mutagenesis on 4 ATF1 (200,220,250,270)
 * 4) Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis