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

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(Mutagenesis)
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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 12:06, 10 August 2006

to do

  1. GC work with new smell samples
  2. Miniprep all LCs/glycerols
    • Make 10 biobrick named glycerols and send for sequencing
  3. Make LCs of IK transformants
  4. plate reader test
  5. PCR pchBA with 5% Betane
  6. site-directed mutagenesis on banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers
  7. 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

  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
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