cRACE problem

Don Walthers gene_jockey26 at yahoo.com
Wed Jun 9 23:08:48 EST 1999


Hello,
I used the Trizol reagent to isolate mRNA from Myxococcus cells. I designed
a 26bp phosphorylated primer for 1st strand cDNA synthesis that anneals
about 150bp upstream of the ATG. My nested primers anneal just upstream of
the cDNA primer and each have a few bp of spacing between. I used Mlu RT
from NEB and T4 RNA ligase also from NEB. I get a 1st step PCR product that
spands a few hundred bp as expected and a 2nd step PCR pruduct that is a
discrete band. When I clone the product (Topoisomerase cloning) and sequence
it, my junction fragment looks somewhat unusual. Instead of a junction
fragment that represents the beginning of the message and the 5' end of the
cDNA primer, the junction contains sequence that looks like the beginning
end of the message but it is ligated to sequence upstream of the cDNA primer
(further downstream relative to the coding sequence). In other words, the
junction fragment contains extra sequence, between the message and cDNA
annealing site. The ends of my cloned sequence are represented by the 2nd
step primers as expected. Any ideas where this extra sequence came from? Is
it possible that topoisomerase ligated two single strand PCR products
together and to the topo cloning vector and it was just chance I picked a
clone that almost looks like a cRACE result? Thanks for any suggestions.
--
Don Walthers
Microbiology
University of Idaho
gene_jockey26 at yahoo.com





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