Church hyb solution questions

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Mon Feb 20 06:11:02 EST 1995


In article <1995Feb18.092832.6739 at ucbeh>, converrl at ucbeh.san.uc.edu writes:
>     I had some questions about church hybridiation solution:
>
>1. What is the original reference? (I have tried like anything to locate it & I
>cannot)
>
>2. Is the method of calculating the optimal hybridization temperature the same
>as for the traditional hybridization mixtures?

The original reference is : Church, G. and Gilbert, W. 1984. Genomic
sequencing. PNAS USA, 81, 1991-1995. While hybridization buffer remained
unchanged, the wash protocol was subsequently modified in many different
ways ( sorry, don't have all these references ). The most useful, from my
point of view, recent version is found in Hauge, B.M. and Goodman, H.M.
Genome mapping in Arabidopsis, in: Methods in Arabidopsis Research, Koncz,
Chua and Schell, eds. World Scientific Publishing Corp. 1992, p.191-223.
I hybridize in the original solution everything including oligonucleotides,
but washing conditions can be different. When I need to reproduce other
authors conditions, I just use SSC-based buffer for stringent washes, as
indicated. It is also possible to include a final TMAC wash into this protocol.
Since most of filter bound ( not DNA-bound ) radioactivity is removed with the
first wash in 0.1 M phosphate-1% SDS at room temperature, one may safely go 
on with any other wash protocol, if one wishes to reproduce any specific 
conditions ( including temperature calculations, where necessary ).

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
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