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

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(Mutagenesis)
(to do)
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==to do==
==to do==
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#GC work with new smell samples
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#GC work with new smell samples -'''rescheduled for 8/11'''
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#Miniprep all LCs/glycerols
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#Miniprep all LCs/make appropriate glycerols/sequence
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#*Make 10 biobrick named glycerols and send for sequencing
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#*Remember to Make 10 biobrick named glycerols and send for sequencing
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#Make LCs of IK transformants
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#Make LCs of IK transformants in pm
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#plate reader test
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#PCR pchBA with 5% Betane
#PCR pchBA with 5% Betane
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#site-directed mutagenesis on banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers
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#site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers
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#clone WGD into pseudomonas shuttle vector
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==Indole KO Strain==
==Indole KO Strain==

Revision as of 13:15, 10 August 2006

to do

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

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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