Reamplifying my PCR product - (Mar/02/2006 )

hiya! i was wondering if anyone can help me?

i am trying to reamplify my PCR products but i can't seem to get it right. i have tried altering the dilution of my PCR product to 1:900 and i have also tried altering the annealing temperature. however, i still can't seem to get a clear distinct band as i can from my initial PCR product, the samples either appear to smear down the lane or don't run from the well at all.

has anyone ever tried this before? what worked best?

thanks in advance for any help. i really appreciate it!

I've always had trouble doing this. I think small amounts of shorter products selectively amplify better than the desired product. If you can, use nested or semi-nested amplification for the second round.
You might find that running the initial product on a gel and pushing a pipet tip into the correct band would be a good way to add template to a second round of amplification.

thanks for the reply phage434! unfortunately this doesn't really solve my problem. i am trying to organise a pcr practical for undergrad students. the aim of the practical is to amplify DNA from two patients, one with normal DNA and another with mutant DNA. once the students have amplified the DNA from both patients they will then do a taq digest to find out which patient has the mutation. unfortunately, we are running short of DNA samples and so we need to find a way to use the amplicons as pretend DNA for the students...if you understand what i mean!

we have tried diluting down the amplicon sample and adjusting the annealing temperature but we still can't get a distinct band on gel electrophoresis. we can get a very faint band but there is a lot of smearing and this is not what students would expect when they think they are amplifying DNA.

would you be able to recommend something else?

Can you still do PCR from your original samples? It sounds like you may have degraded primers, but it is hard to know without knowing if you original PCR works. How are you storing and resuspending your primers? Many other things could be wrong. Are the dNTPs still good? Can you do other PCRs with the same reagents? Is your water quality good?

Have you gel purified your initial PCR product? Recall that anything amplified at all during the first round (spurious products included, to whatever degee -- even a vanishingly small amount not visible on your initial gel) will, by definition, contain your primers on the ends.

During the second round, these will amplify equally well as your intended target, resulting in a smear.

Alternatively, do the students actually need to amplify the entire original sequence to observe the intended results? If not, you could synthesis some primers that the students will use that extend a bit into the amplified DNA, so the 3' end of the student primer actually requires the correct template to anneal. So, you'd be setting up a situation like this (+'s and *'s indicate primer bases, _'s indicate amplified DNA):


 ++++++++++_______________________++++++++++ student PCR
**********___________________________********** original PCR

yeah, i can still do pcr from my original samples using the exact same reagents. i am storing my primers at -20oc, they were made up my supervisor and i completely trust they are ok. i am using ultrapure sterilised water (18mg), fresh everytime.

from what you have said, it could be my dNTPs, i have had them for a while and they could now be degraded from when i originally started to use them. i think i will try using new dNTPs and new primers and see if that makes a difference.

i have been trying to think of all the variables that could be affecting my results and could it be the number of cycles i am using? in your experience does this make much difference?

i will try my experiment tomorrow using new dNTPS and Primers and let you know how it goes!

thanks

thanks for your reply homebrew! its given me something to think about. i'll discuss it with my supervisor and see what he thinks.

i have been able to run my original sample on a gel and produce a really clear single distinct band on the gel for both mutant and normal patient. i have also been able to taq digest the mutant sample to prove that it was the mutant sample so i know that the amplicon which i am trying to reamplifying contains the 'right' DNA.

like i said in my previous post i will try it again with fresh dNTPs and primers and see if there is any difference.

but any other hints or tips in the mean while will be greatly appreciated.

It seems unlikely that it would be one of your reagents, given that the first PCR works repeatedly, with what I assume are the same reagents.

I would run my first PCR out slowly on a long-bed gel, slice out the band of interest, purify it away from the agarose, and try using that as template for the second PCR. If the second PCR doesn't work well enough with that gel-purified template, I'd pick some student primers that each contain a few (two or three) 3' bases specific to the DNA amplified by the first round, as I described schematically above.