about transformation of DNA into E.Coli

Alexander Kraev kraev at bc.biol.ethz.ch
Wed Aug 14 04:37:22 EST 1996


>

        Glad that you were partially successful. But to improve your
tranformations.

1. Pre warm your plates for longer times. 15min, isnt good enough.. try
atleast 60min.. the plates should be really warm and dry.

2. Initally incubate your DNA with Cells for 5min till you get  the hang
out of your method.

3. Clean your ligation mixes, since the ligation buffers usually kill 
your
cells.<

Sailesh, could you please post which E.coli strains you have actually 
used? My point is, we have found in the past, though not published, that

1. Some strains, e.g. XL-1, are pretty insensitive to the length of
the heat shock
2. Some strains cannot be efficiently transformed by the Hanahan protocol 
but can be electroporated >100 fold more efficiently (SURE)
3. Some strains give similar efficiencies with electroporation and the
chemical transformation
4.Some strains are particularly efficiently electroporated, e.g. MC1061
and DH5alpha. 
 The tendency is that the strains with fewer mutations and generally
more "vigorous" are better electroporated than chemically transformed,
and various relatives of the DH5/XL-1/AG-1 (though not SURE) are equally 
efficiently transformed by both methods. What is your experience?
 











Good luck with more efficiencies.



Alexander Kraev, PhD                     Internet:    
kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
> Swiss Federal Inst. Of Technology           FAX:      0041-1-632-12-13
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> CH-8092 Zurich                                     /Sasha/kraev.html





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