High Efficiency Transformation (libraries)

Rafa Maldonado Rafael at genetics.med.utah.edu
Wed Feb 16 21:17:15 EST 1994


In article <CLBrn5.1CD at usenet.ucs.indiana.edu>
jgraham at bronze.ucs.indiana.edu (the End) writes:

> Has anyone actually achieved transformation efficiencies near the 
> 10(9)/ ug reported by Hanahan ? This would require a CsCl purified 
> spec quantitated control plasmid.
> 
> Are there any variations reported in the last several years that 
> are working well ? 
> 
> I am intersted in making libraies from a relatively inefficient 
> ligation.
> 
> Thanks much,
> 
> Jim
> J. Graham

Yes, there are some improvements to Hanahan protocol. Check:

Okayama H, Kawaichi M, Brownstein M, Lee F, Yokota T, Arai K (1987)
High-efficiency clonning of full-length cDNA; construction and
screening of cDNA expresion libraries for mammalian. Methods Enzymol.
154, 3-28

and my favorite:

Inoue K, Nojima H, Okayama H (1990) High efficiency transformation of
Escherichia coli with plasmids. Gene 96, 23-28

The last one is very good. They found that some compounds oother people
used in transformation buffers are inhibitors of the transformation
(!!!???). One critical point is the temperature of incubation of the
bacteria: 18 C is the optimum (!!!). Also, the heat-shock time is
reduced to 30 s. It works very well, in my hands and with DH5a. The
frequencies usually are around 4.10(8); 10(9) only in the good days:
it's really difficult get that frequency!
On the other hand, have you tried electroporation? Maniatis's protocol
gives good transformation frequencies too; I have read somewhere that
MC1061 is the best for electroporation.


Good luck!

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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