Primer Extension Sequencing
bressler at u.washington.edu
Wed Mar 6 16:30:03 EST 1996
Dear Sequencing Wizards,
I would like to sequence primer extension products. We are near the 5'
end of a gene but would like to get to the 5'transcriptional start site by
using primer extension. I am looking for suggestions how to sequence the primer extended
product. After kinasing and labeling the oligonucleodtide
with gamma 32-P ATP and then hybridizing the labeled oligonucleotide to
approximately 50 ug RNA-followed by an overnight incubation and then
precipitation and here is where I am asking for advice
prepare a RT mix but if I also want to sequence the product what is the
best procedure. Do I add the ddNTPs to the RT reaction or do I do second
strand synthesis reaction after the RT reaction first and then
precipitate and sequence the double stranded product?
Please reply by E-mail. I thank you very much
Steven Bressler, Ph.D.
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