The Case of a Missing Band: PCR Issue
Posted 22 May 2010 - 05:17 AM
1. My first question is, would this approach be better ( in terms of sequencing) that just amplifying the whole 3kb gene with a taq polymerase that can do that?
2. I was already able to optimize the PCR conditions for primer sets 2 and 3. The first time I ran primer set 1, using two different DNA samples and a tempearture gradient, I got a band at 60C for sample 1, and for 50, 55 and 60C for sample 2. Is this possible? The two DNA samples come from the same species but different isolates.
3. I tried several conditions after that, but did not get that band anymore. I even prepared a new primer solution but still did not get any band. How could this happen?
4. What other approaches can I do to amplify my target gene for sequencing? (I also tried degenrate primers but this did not work).
Posted 22 May 2010 - 09:49 AM
Do you already know the sequence? if yes, why do you want to use degenerate primers?
Posted 22 May 2010 - 10:17 AM
Posted 23 May 2010 - 05:43 PM
Posted 25 May 2010 - 06:57 PM
am pudin jus newly joined this forum.
my pcr pdt is 1.2kb, fp is 40%, 67C& rp is 60%, 73C gc content and Tm resp. , am nt able to amplify my template. i could find primer dimer and non specific amplification of 500bp. i tried tm of 55, 57, 59, 60, 61, 62,63,64, 65.
help me out plz,,,,,