How do you prepare PCR stock solution to minimize pipetting - (Mar/29/2005 )

Hi Shiva :
I tried your advice, the results were good for the DNA that extracted from blood by manual method (salting out) that I used as a control for my new DNA that extracted from Buccal swab by Kit..but all the buccal samples failed (of the same run)..have u any idea ?? Sooooorry I didn't tell you that I used different DNA at the begining,, coz I thought it may not infulence the results..

Thanks a lot Shiva

Hi all :
the problem that i found in amplifing certain gene from buccal swab extraction ,,, solved easily by Whole Genome Amplification (WGA) ...
thanks all

hi everyone.

im having a problem in pcr... i did my pcr mix in 50 ul and it always gives a perfect band but now i need to do pcr mix in 25 ul and of course i cut everything to half dntp, taq except for primers and dna which is 0.5ul but after gel running there is no band. and im using the same dna as in 50ul pcr mix. i did my troubleshoot but due to my inexperience in pcr i found nothing. i used TAE buffer if that helps. plz help...

hi everyone.

im having a problem in pcr... i did my pcr mix in 50 ul and it always gives a perfect band but now i need to do pcr mix in 25 ul and of course i cut everything to half dntp, taq except for primers and dna which is 0.5ul but after gel running there is no band. and im using the same dna as in 50ul pcr mix. i did my troubleshoot but due to my inexperience in pcr i found nothing. i used TAE buffer if that helps. plz help...

thanks for ur reply...do u mean that maybe dntp, taq, etc that may caused the problem is it? okay ill try to repeat it with some modification in it. thanks.

I agree with mdenko

the proportions of reagents MUST remain the same. if it is too difficult to pipette a smaller volume, do a 1:2 dilution (in the buffer they are already in) and that will allow you to halve the volume in the PCR without trying to pipette 0.2 or 0.1 ul or something silly like that

another thing, when you say you use TAE buffer, what exactly are you putting it in? that is a buffer for electrophoresis, not for PCR or for resuspending DNA...where are you using it?

Am having problem with a trivial PCR so much so that I have started doubting along with my bad Karma, the proper mixing of components. Will it help to mix water and buffer properly before adding other components? What could be the problem assuming I am doing the the work properly if there is no problem with 10X buffer, dNTPs, template, taq, primers, water and you are getting no amplification. Some voodoo magic??

In general I add the enzyme last, on the theory that I don't want to expose it to un-buffered conditions. For your PCR problem my suggestions are to try again with gradient PCR, varying the annealing temperature. If you know your insert has high AT, try lowering the extension temperature (65 C). If you know your insert has high GC, try adding 5% Betaine. You could try increasing the Mg++ concentration of your buffer by 1 mM. When you get tired of this, redesign your primer. I usually skip all the stages except the first and go directly to a primer redesign. If you are working intensively on a construct, you may have other primers which can paired with the non-working ones to figure out which primer is the problem, and redesign only the troublesome one.

Calvin, even if you are using the same source for water, the pH may have shifted?

I am grasping, as it seems you have tried all the 'easy' fixes. have you spec'd your template? what I mean is, are you sure the DNA is good? have you tried a control reaction, using a different set of primers, with conditions that have worked for you in the past on the same DNA template? there is a possibility that your thermalcycler needs repair, but I would take that as the last resort

are you even getting primer-dimers, or non-specific product, or anything at all on the gel?