Primer Extension Sequencing

[Steven Bressler]

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.