User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/15

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Objective

Competent cell prep of E. coli ER2566 using Hanahan (1983) JMB 166:557-580 method, modified as follows:

  • The final Transformation Buffer is: 45mM MnCl2, 10mM CaCl2, 100mM RbCl, 3mM hexamminecobalt(III) chloride, 10mM MES (pH 6.2), 7% DMSO, 75mM DTT, 10% glycerol. This is the TB in the paper + glycerol, for freezer storage.

Bench work

  1. The competent cells were prepared by Kay Wang and Donald Lee. They were quick-frozen prior to transformation.
  2. Test transformation with pKSeAATHis by James Page and Kay Wang with DH10B, ER2566, and W945T1-2λ-

Results

  • Low transformation efficiency of ER2566 - must be re-confirmed
  • Kathryn Muratore 15:37, 19 July 2011 (EDT): re-confirmation (by Tamra Fisher) shows low, but non-zero transformation efficiency. We are trouble-shooting the competent cell and transformation protocols, but this prep should work for transforming pure plasmid for expression.
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