LA-PCR

unknown at dl.ac.uk unknown at dl.ac.uk
Tue Dec 7 11:47:16 EST 1993


Dear colleagues-on-the-net,
I have a question regarding RT-PCR. Currently (for the last
couple of months, that is) I am trying to clone a number of cDNAs
using the `ligation-anchored PCR' method, as described by Troutt
et al., PNAS 89, 9823-9825 (1992). I do get lots of PCR product,
but they are only very small (around 250-300 bp) instead of say
800 as expected from sequence data. The method is designed to use
T4 RNA ligase to add an anchor primer to the 3' end of the 1st
strand cDNA (i.e. the 5' end of the mRNA), and perform PCR using
the complimentary sequence as one of the primers. Could anybody
give some hints as to what might cause the truncated product to
be formed? Probably something obvious, that I can't see from
staring too hard. Btw, the RNA seems to be intact, judging from
its pattern on an denaturing agarose gel. Grateful for any tips,
regards, 

Han Schilthuis, Utrecht, the Netherlands 
E-mail: hl49 at hubrecht.nl



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