39 replies to this topic

[xabiarias]

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.

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[Ameya P]

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....

[xabiarias]

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?

[Zach T]

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.

Edited by Zach T, 04 March 2011 - 11:47 AM.

[xabiarias]

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?

[Adrian K]

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

[Adrian K]

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

[Ameya P]

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???

[xabiarias]

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!

[Ameya P]

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!

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.

[xabiarias]

I used QIAmp and flexigen (both from Qiagen) when everything was ok and the amplifications were succesfull. Currently, I am obtaining precipitation in almost all of the samples. One researcher, which has been working on this for a long time (in othe city), has suggested to me that optimal primer concentration has to be determined every time new primers are bought; he believes that the final theoretical concentration is not enough and has to be experimentally determined. He also states that the order in which the DNA, primers, water, buffer,etc are added is vital in the effyciency of this long PCR reaction (I was astonished whe he told me this issue)!!

[Ameya P]

I used QIAmp and flexigen (both from Qiagen) when everything was ok and the amplifications were succesfull. Currently, I am obtaining precipitation in almost all of the samples. One researcher, which has been working on this for a long time (in othe city), has suggested to me that optimal primer concentration has to be determined every time new primers are bought; he believes that the final theoretical concentration is not enough and has to be experimentally determined. He also states that the order in which the DNA, primers, water, buffer,etc are added is vital in the effyciency of this long PCR reaction (I was astonished whe he told me this issue)!!

I am equally astonished.... as I read your post...

Primer concentration may be an issue if the scale of synthesis if different. But on most occasions, a lab orders primers synthesized on the same scale as all other primers. Check the scale of synthesis for the sets of primers you have. But its unlikely they will be different.

Well, everybody has an order of adding ingredients to a reaction, but with all due respect to the researcher, I dont think it should affect the efficiency of the PCR .

Although, somewhere we all know that "PCR master mix making" is a holy ritual and must be done as per our own SOPs , the end result in the tube, before it goes into the thermal cycler is the same.... irrespective of the order in which you add the ingredients.

I follow a simple rule, water first and buffer before the polymerase. Others dont matter.

[xabiarias]

Thank you for your advices..The primer scale are the same as I bought them bymyself from Invitrogen.

This time I've modified primer concentration and just realized that the precipitation still appears inside the PCR tubes after the PCR reaction (I have to chek them in an agarose gel that I am running). Even this time I have followed the intructions of how the components have to be added! :S Now I am going to try a PCR adding less DNA and DMSO following some advices...I only want to get rid of these precipitation; the amplification of my bands doesn't really matter now!...

A comment from Zach T would specially appreciate because he experienced the same smeary bands in a long PCR. Anyway, comments/advices, from anybody, would be very very appreciated.

I'll keep you informed with the next PCR!

[Adrian K]

Do you experience any reduction of volume of your PCR tube after the PCR reaction?

I suspect that your PCR tubes have some leakage which caused evaporation and thus the precipitates happen. Are you using new batch/brand of PCR tubes lately?

Also, I suspect as well the Qiagen elution buffer (Buffer AE, or carryover of Buffer AL) "might" had interfere with your SequalPrep kit (probably with the 10x enhancer)and caused some precipitates. The SequalPrep kit does mentioned not to use TE buffer when performing your elution of DNA, but use distilled water instead.

By combining the factor of tube leakage and buffer interference, and considering the fact that your PCR total volume is only 20ul (based on manual), there comes my precipitation forming hypothesis.

I do had experienced with ~10kb PCR, but I didn't use invitrogen kit. But lets first find out whether is your tube's "capping" problem as a start.

P/s: Buffer AE: 10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0.

[xabiarias]

It seems that no reduction in the volumen occurs throught the PCR reaction (but I haven't measured them). The PCR tubes are new and also autoclaved. I usually arrange a final PCR volumen of 25 ul and I forgot to mention that I've been performing the DNA elution from the kit with nuclease free water!