Cloning PCR products

Ken Soderstrom soderstk at ucs.orst.edu
Tue Dec 31 13:21:30 EST 1996


I made an oligo(dT)-primed lambda-ZAPII cDNA library from amphibian RNA.  

Using degenerate primers I've been able to amplify (with Taq) ~700 bp of 
the  ~1100 bp product that I'm interested in cloning.

Using this 700 bp fragment to screen 250,000 pfu I isolated only one
non-full-length clone: the 3' ~800 bp.  

I've been able to amplify (using Pfu) the 5' end using an internal 3'
degenerate primer and a 5' primer designed to amplify the sequence from a
mammalian species: this primer is ATG + four mammal codons.  

The 700 bp amphibian Taq fragment and the mammalian sequence are >90%
conserved at the aminoacid level.

I would like to avoid a brute-force screening of this library if possible.

I would like to ligate my 3' clone and the 5' Pfu product and get on with
making a cell line.  

Will I be criticized for a potential error in the four 5' codons?  Given
that the mammalian primer resulted in amplification, I expect that the
sequences are very similar.  Also, I don't anticipate that changes in the
extreme 5' portion of the protein will have functional consequences.

Any opinions?

Thanks,

-- 
Ken Soderstrom
soderstk at ucs.orst.edu




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