User:Tk/Notebook/MF-xfm/2008/04/13: Difference between revisions

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(Autocreate 2008/04/13 Entry for User:Tk/Notebook/MF-xfm)
 
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==Entry title==
==Transformation results==
* Insert content here...
* E. coli transforms at 400 colonies
* No MF transformants on any plates
* Frozen transformed stocks from A and B grew well on the same plates
* C did not grow, nor did wt MF
* yeast (or other contaminants) on two plates.  This is likely from the ice bucket water.
 
==Remaining speculations==
* wrong DNA sequence
* too low a cell density
* hold on ice for 10-30 minutes prior to electroporation
* recovery wrong
** recovery at low temperature (perhaps in the cuvette)
** osmolarity of the recovery medium is wrong
* Test higher osmolarity of electroporation buffer than wash medium
** some used 0.5 M sorbitol and 0.5 M manitol, 10% glycerol
** some buffers had Mg++, but we can't use it due to transposomes
 
==DNA sequence analysis==
* PCR from A/B strains
* PCR from transposome mix
* PCR from transformed E. coli
 
==Good controls==
* E. coli transformation
* growth of MF tetR strain
* measurement of CFU in transformed input




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Revision as of 21:48, 19 June 2008

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Transformation results

  • E. coli transforms at 400 colonies
  • No MF transformants on any plates
  • Frozen transformed stocks from A and B grew well on the same plates
  • C did not grow, nor did wt MF
  • yeast (or other contaminants) on two plates. This is likely from the ice bucket water.

Remaining speculations

  • wrong DNA sequence
  • too low a cell density
  • hold on ice for 10-30 minutes prior to electroporation
  • recovery wrong
    • recovery at low temperature (perhaps in the cuvette)
    • osmolarity of the recovery medium is wrong
  • Test higher osmolarity of electroporation buffer than wash medium
    • some used 0.5 M sorbitol and 0.5 M manitol, 10% glycerol
    • some buffers had Mg++, but we can't use it due to transposomes

DNA sequence analysis

  • PCR from A/B strains
  • PCR from transposome mix
  • PCR from transformed E. coli

Good controls

  • E. coli transformation
  • growth of MF tetR strain
  • measurement of CFU in transformed input


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