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

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)!!

xabiarias on Sat Mar 5 10:48:51 2011 said:

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.

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!

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.

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!

xabiarias on Tue Mar 8 17:17:45 2011 said:

Try to run a comparison between autoclaved and un-autoclaved tubes. From my experience, my autoclaved PCR tubes tends to have the issue of leakage and cause volume reduction, and this is the reason I never use autoclave tubes anymore. Not sure whether is this the case for you.

Why not try to email invitrogen and see what their tech have to say?

Pretty interesting not using autoclaved PCR tubes. I have to probe it. I actually talked to Invitrogen's tech and they provided me a new kit (in the beginning I thought that the problem was the polymerase!)

My PCR tubes were certified 100% virgin polypropylene and are lot tested and certified to be free of RNase, DNase, and Human DNA ..
I'm using PCR Tube with Flat Cap 0.2ml, Labcon <3936-500> Price RM180 @ ~USD45 per 1000pcs.

I was wondering why would you have to autoclave your tubes? Can share?

You don't autoclave your filtered tips, do you?

Xabi,

I too use un-autoclaved tubes for PCR and haven't had any issues so far... Mine are Axygen (PCR-02-C).
I dont use autoclaved tips either. They have filters though.

Adrian,

I dont think autoclaving the tube might be a reason for the precipitate.

I've obtained the precipitate again in conditions without DMSO and enhancer A (needed for the correct amplification). So we can rule them out. I've just prepared a PCR reaction with Taq Platinum Buffer plus Mg plus dNTPs instead of SequalPrep Buffer (and of course with DNA, primers, polymerase and water). We'll see if it works out!