Protocol Online logo  Sequencing of scarce real-time amplicons -
(Jun/18/2009 )  Dear colleagues,  does anyone have experience on
a succesful sequencing of a scarce Taqman real-time PCR product?
How have you managed to do that?  I have a very little amount
(Ct-value 30-35; primary volume 25 ul) of a small (88/89 bp)
amplicon, that is very challenging to sequence. Almost all of
the original sample RNA has already been used for different
attempts. Unfortunately the sensitivities of my conventional
PCRs are insufficient, so I am not able to amplify my target
with them. I have tried 1) Qiagen PCR purification kit 2) Qiagen
gel extraction kit 3) TA-cloning and 4) ExoSAP purification, all
followed by sequencing attempt with the real-time primers that
gave the positive result. I have also tried to amplify the
amplicon with the real-time primers but with conventional method
to get more template; this did not function.  What could I try
next? It would be utmost important to find the sequence since
this is the first fi