RTPCR

bina roy roybina1 at yahoo.co.in
Wed Sep 25 08:18:24 EST 2002


Dear All,

I wish to know that how you can optimize the PCR conditions (eg. # of
cycles etc.) for a particular product you are amplifying. Do you have
to repeatedly run the PCR product in a gel and determine it.

My second question is that if I want to determine the best possible
forward and reverse primers for a particular gene (if I am unable to
find any published ones for that gene, nevertheless the sequence is
known), what are the good softwares for that. I am familiar with
Primer 3, but I want to recheck the primer sets again in a separate
program to prevent the possibility of primer-dimer formation. How am I
supposed to know if the chosen primers worked for me or not. By size
of the product after running on a gel?

A third question concers about choosing primers for a given set of
genetic markers. Do I have to plug in the marker (like D1S228) say in
PUBMED to know its sequence and then design a primer from there. Is
there any link that might help me to know if there are any primer sets
available as information.

Thank you all for helping me out.

Bina.



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