5 replies to this topic

[sihyunie]

I PCR'd out 2 fragments from 2 different plasmids.
After few failed attempts to fuse these two fragments, I went back to the beginning to figure out what's wrong.
I took the gel purified fragments as templates for another round of PCR with the same primers.
To my surprise, there was no amplification for one of the fragment.
I know that it's not the gel purification, because the other fragment was amplified by the second round PCR.
I can't really think of any reason why this one fragment is giving me a problem (and nobody else in the lab seems to have an answer either).

[NemaToStella]

sihyunie, on Mar 16 2010, 10:24 AM, said:

I PCR'd out 2 fragments from 2 different plasmids.
After few failed attempts to fuse these two fragments, I went back to the beginning to figure out what's wrong.
I took the gel purified fragments as templates for another round of PCR with the same primers.
To my surprise, there was no amplification for one of the fragment.
I know that it's not the gel purification, because the other fragment was amplified by the second round PCR.
I can't really think of any reason why this one fragment is giving me a problem (and nobody else in the lab seems to have an answer either).

Hi,
what how much of the PCR product that can't be re-PCR'd did you use in the unsuccessful PCR reaction? Could you be using too much template? Also, have you considered sequencing this PCR product (if it's not too small that is)?

[sihyunie]

I used 1ul out of 50ul PCR reaction.
I'm not sure if it's because of too much template because the other 2nd round PCR worked.
I'll probably sequence it, but I'd be amazed if a PCR product of expected size wasn't of the correct sequence (although all evidences point towards incorrect PCR product).

[Maddie]

Did you get a smear? If you did, them you definitively used too much DNA.
I'd dilute my first PCR product quite a lot for the 2nd amp.

[merlav]

If your primers are well design and have 1 band only you don't need to gel purify the first amplification.  doing this your are loosing dna material, time and money.  For a better result in a nested pcr the second primer set should be near the center of the 1st amplification.  The least product you see at the first amplification the better amplification in the 2nd round should be see.  If have a band in the first amplification dilute the product at least 1:5 (I prefer 1:10).  For secuencing I recommend purify the reaction with a centricon filter or a filter base purification kit (I have seen a better yield).  Sometimes the gel purification yield is too low.  The last recommendation don't sequence until you are sure that you have a good amplicon.

Edited by merlav, 25 March 2010 - 05:18 AM.

[swanny]

Could you please clarify for me exactly what you want to do? Are you trying to make a new construct of the two PCR products fused together?

What kind of sequences are you using for your primers?

If you're not getting a single band from your first-round PCR, I'd suggest you try to do so. Change the annealing temp, try a touchdown PCR. Then when you have a single band, don't bother with gel purification, just use a small amount (0.5 - 1 ul) of the first reaction in the second round.