Best strategy from peptide sequences to cDNA clone?

Eric C. Anderson anderson at pharmdec.wustl.edu
Tue Oct 11 00:04:51 EST 1994


In article <3744oi$6q6 at news.iastate.edu>, yqhuang at iastate.edu () wrote:

> This is a cloning strategy question:
> What's the best strategy from peptide sequences to the cDNA clone?
> 
> As we all know, we have at least 4 strategies to clone the cDNA.
> 1. degenerate oligo probes;

i used a degeneral oligo probe to PCR clone a portion of a C.elegans gene
based on orthologous sequences from other organisms.  the best results i
had were using primers where 3 or 4 base degeneracies were made using
inosine.  this introduces some other concerns (see the IRL Press book "PCR:
A Practical Approach" for more info here), but it seems to make things work
better than using mixed 3 and 4 bases.

> Is non-radioactive probe good for library screening? 
> If yes, then what labelling is good or appropriate? biotin? digoxingenin?

i have used the Beohringer Mannheim Genius (digoxigenin) system for
non-radioactive labelling of Southerns and dot blots with great success and
two other people in the lab have used it for library screens with good
results.  it seems to work very well and i highly recommend it.

good luck,
eric

-- 
*************************************
*Of course it's not true...         *
*But let's make the bastard deny it!*
*                        LBJ (1948) *
*************************************
eric c. anderson                                
anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110



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