The Case of a Missing Band: PCR Issue - (May/22/2010 )

Hi all, help me sort out my results in PCR. I am trying to amplify a putative ~3kb gene. I designed three primer sets that I expect would give three overlapping sequences that I can put together.

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).

Please help.

If your purpose is to sequence the 3kb fragment, I would suggest that you amplify the whole region in one PCR reaction and design one or two internal primers to sequence it.

Do you already know the sequence? if yes, why do you want to use degenerate primers?

I agree with pcrman -- 3 kb should be a piece of cake for any polymerase. Your best approach if you're not going to clone the fragment is to use Taq and your two outermost primers, recover the amplicon from a gel, and send the recovered DNA for sequencing in several aliquots using the primers you used to amplify it, plus the other internal primers you designed. If you're going to clone it, use a high fidelity polymerase, and send the recovered plasmid for sequencing, again using all primers.

Thank you for the inputs. As for the degenerate primers, I did that before I got hold of a sequence sent to us by another party. Regardless of the approach, my problem is that the first primer (amplifies the beginning of the gene) is not working. Should I just redesign?

hi all,

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,,,,,