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?
It may not be the contamination. Most likely the ppt can come from your pcr buffer since it is the only chemical with high salts.
First you need to check the starting DNA material is fine before amplification. You can load 50nanogams of DNA on a gel and make sure that there is no smear. Once it is fine then changing the pcr buffer may solve your problem.
Thank you. The DNA seem sto be ok in the gel and in the spectroscophic characterization. I've tried different primer concentration and precipitate still apears even without DMSO and the Enhancer. Experiments still performing!
If your pcr buffer and starting DNA template are fine and still if you see ppt after pcr it must be the ppt of EDTA. By any chance ru using TE to re suspend the DNA or primers? If so incase the TE become old then there will be chance of change in pH. If pH reaches to below 8 by any means then EDTA will be precipitated. Please make sure all your reagents have pH around 8.2. In that case re suspending DNA and primers in water may solve your problem.
We met the same thing. It's BSA's precipitation. We also don't know why. Are there any expert can help?
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