difficulty in Primer walking - (May/16/2006 )

Hai everybody,

i am trying to tail my RT product(cDNA) with dATP / dCTP using Terminal Deoxynucleotidyl Transferase for primer walking inorder to sequence the downstream elements of a negative sense RNA virus, but i am facing difficulty in getting bands in my PCR product(wherein i use a known forward primer and oligo dT/ G anchor as reverse primers) can anybody help me with some tips as to how i can improve my technique to get good bands or atleast good smear from the PCR product (i have used many controls to rule out the possibilties of any problem with the RNA i am starting with and also the cDNA and also the PCR technique)

PAAPU

Two things to try:
Use TTTTTTTTTTTTTTTTTT{A,G,C} or GGGGGGGGGGGGGGGGGG{A,T,C} as the primers, which will prime more specifically.

Try replacing the TdT addition with a ligation of a specific primer (must be 5' phosphorylated) using T4 RNA (not DNA) Ligase. Make sure you add ATP to the buffer or use the T4 DNA ligase buffer.