PCR probe

John Stiles stiles at uhunix.tmc.edu
Thu May 12 20:16:52 EST 1994


In article <2qrf7u$emm at news.u.washington.edu> flavio at u.washington.edu (Flavio Solca) writes:
>Hi netters
>
>I am trying to use a 265 bp PCR fragment to screen a lambda gt11 library. 
>Random labeling this probe works reasonably well but the fragments that 
>are produced are a tad small. I hadproblems in getting a signal on my 
>filters.
>
>Does anyone know a way out?
>
>I was thinking of generating long concatemers using high conc. of 
>repaired PCR fragment and T4 ligase. I was hoping that by using this mix 
>I would get some longer pieces. Did anybody use this approach?
>
>I would for sure appreciate comments,ideas and protocols.
>
>Flavio		(Flavio at u.washington.edu)

Just to confuse the issue I'll give my favorite method.  Kinase both PCR primers in one reaction, EtOH ppt, dry and add PCR reaction stuff and go for it.  I don't like adding a 'bit' of labeled dNTP because PCR requires a lot of dNTP so, although you get a lot of counts the SA is rather low.  I've used kinased PCR frags to screen libraries and it works well.  Good luck.

John Stiles
stiles at uhunix.uhcc.hawaii.



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