ful length CDNAs

Ralph M Bernstein ralph at ccit.arizona.edu
Thu Jan 19 09:40:57 EST 1995


To 
Jon Nakamoto
Clinical Instructor of Pediatrics/Endocrinology, UCLA
jnakamot at pediatrics.medsch.ucla.edu

OK, John, there is a nifty new procedure out just published in Nucleic
Acids, and I can give you the exact ref if you need it.  it utilyzes a
single primer and random priming.  the article was called "race no more...an
alternative to 5'race ...cloning strategy.."  he used a single primer (a
sense) in 50 rounds of lineal amp, then eluding the ss dna band (after 50
rounds ss dna can usually be visualized) then re-amp with the same primer . 
pcr/taq/whatever accidentally adds the primer onto the 3'end of the
transcript/ss dna and logarithmic amp then occurs.  he has had seemingly
wonderful success with several primers, and really is amazing.  

although, traditional 5' race is notoriously bad at amplifying large 5'
ends, eg over 1 kb.  i know that most kits say up to 3, but in practical
experience, 2 kb is actually pushing it---and since i dont remember your
orig posting, i dont know if you need to get a large 5 piece---the technique
i mentioned was used on about .3 or .5 kb sizes.  any specific questions,
though you seem to have race down pretty good, just email me.  Ralph
Ralph M. Bernstein
Dept of Micro/Immuno
University of Arizona
Ph: 602 626 2585
Fx: 602 626 2100



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