Smear in long distance PCR - (Mar/02/2011 )

Hi Guys,

I´ve been performing a long PCR determination of coagulation factor VIII intron 22 inversion (with SequalPrep kit from invitrogen (A10498)). In the beginning everything was fine and I could manage to amplify the corresponding bands (of 12 kpb in wild-type or 11 kpb in inversion positive patients). But suddenly an strange thing happened: in almost every PCR I tried, I got smeary bands in the agarose electrophoresis. I realized that the smeary weird bands were the consequence of the presence of some kind of white precipitation that appears in the PCR tube after the amplification. The point is that I didn´t change anything in the sample preparation (I previously had done aliquots of buffer, primers, enhancer that I thaw in the moment I need to prepare a PCR reaction). I use the same small room for DNA extraction, prePCR manipulation, postPCR manipulation; the question is, do you thing am I experiencing any kind of contamination? Can sample,primer, buffer or whatever contamination produce the precipitation inside the thermocycler in the PCR? I attach a photo of an agarose gel in order you could visualize those weird smeary bands! Thank you in advance.

Because, you say that you have changed Nothing...

probably its time to change something...

like, How old are your primers? How many freeze thaw cycles have the stocks gone through?
How old is you buffer/ Mg Cl2? Again, how many times has that been freeze thawed?

Try using some one else's buffer. Also, order new primers, if they are quite old (3-5 years). Its just 4 primers... shouldn't cost you a lot....

Hi Ameya,

Thank you for your answer. The primers were not too old (2 month)and in fact I have the same results with new bought ones. The buffer (with MgCl2), enhancer, DMSO and polymerase were from a recent kit. What is more, we have got a very new kit and still experience the same results. I normally do stocks of all these components.

It is very frustating but the strangest thing is the presence of a precipitate after the PCR. This precipitation does not always appears! but when it appears it produces those smeary bands. Do you think this could be a consecuence of some kind of contamination? have you ever seen those smeary bands?

I've actually been experiencing smearing bands similar to your pic using TaKaRa's LA pcr kit, and trying to amplify a 13kb region. Still haven't really gotten it to work right, but I've never noticed a precipitate after the pcr. I think my smearing was caused by high concentration of primers. That precip is still pretty interesting, I would have to think that its contamination somewhere and if its happening with all new primers, etc, maybe your DNA is contaminated or degraded.

Hi zach,
Thank you for you reply. In the beginning I thought it could be the DNA but the electrophoreis and the spectroscophic characterization were normals. I heard that for long PCR the quality of the template has to be very high but my DNA could amplify small exons with Platium Taq polymerase so I guess that my DNA is ok. Do you know why do you obtain those smeary band in your long PCR?

IF thats the case, it might be due to the high salt concentration inside your DNA. How you do purified your DNA?

IF thats the case, it might be due to the high salt concentration inside your DNA. How you do purified your DNA?

I dont have an explanation for the precipitate you are seeing. I had read somewhere on this forum, that MgCl2 must be vortexed and used. I was guessing that probably that could be the source of the precipitate.

Although, you say that the DNA seems ok, you have not mentioned how do you extract your DNA. Is it a manual method? or a kit? Either ways, is that kit too old?

Secondly, does anybody else use the thermal cycler? Are their reactions working fine???

I purify the DNA with QIAmp or/and flexigen kit. The MgCL2 is part of the 10x buffer and nobody else uses the thermocycler. What is more, I have performed the same PCR reaction in other thermocycler with identical results!

xabiarias on Sat Mar 5 09:00:19 2011 said:

So, we can rule out the thermal cycler being the problem. You say your PCR ingredients are fine. That leaves us with the extraction kit. When you say QIAmp and/or Flexigen, is it that both are at your disposal and you just use any one you want? Because, you said the precipitation is not seen all the times.