Hi,
I've been amplifying part of the 18S gene from environmental samples in preparation for Illumina sequencing. We were using invitrogen's AccuPrime PCR kit. Everything was working even though the bands were a little faint... Then, we had some contamination in the neg. control so we got all brand new reagents: water, primers, PCR buffer, MgSO4. I ran the PCR program exactly the same way as before. But suddenly we are getting these very bright smears as shown in the attached image at around 12 kb in size. They are only in samples where we add DNA and not in the negative control. I tried cleaning up the sample with magnetic beads in case it was an artifact in the gel but the smear is still there. Does anyone have any idea what could be causing this? Is it an issue with the gel or with the PCR itself? I am not the only one in the lab to see this; others are also seeing these bright smears even though they are doing different PCRs. I have never seen this before and it's very strange. I would very much appreciate any suggestions anyone has!
Smear due to the presence of inhibitors in samples. Your Primers & Enzymes are unused for the same. Try to Dilute the samples and optimize the PCR cycle.
Dear Jenan,
I would say that something is in excess in your PCR mix. Primers, DNA or Taq.
Could you share your reaction conditions as well.
Thank you for the suggestions...
Reaction conditions are 95C for 2 min, then 32 cycles of 95C for 20s, 56C for 30s, and 72C for 5 min then 1C forever. In each reaction I am putting:
10.05 uL H20, 2.0 uL 10x buffer, 0.8 uL MgSO4, 0.15 uL Accuprime Hi-fi Taq, 1.0 uL of 2 uM F primer, 1.0 uL of 2 uM R primer.
I have not changed anything with the PCR program from before when it was working except to get all new reagents from a new kit and new primers from the 2 uM stocks in the freezer.
I am going to run another reaction on a different cycler, using my old reagents, even though they are contaminated, to see if this band still shows up. Is it possible to get a "bad batch" of reagents? Or that the thermocycler is messed up somehow?
Hii Jenan J Kharbush, since you have not changed the other parameters, from the gel image I feel that you are adding excess amount of template (DNA/cDNA). The stored DNA template can get concentrated during the freeze-thaw cycles due to evaporation. Hence even though you are adding the same volume of template in to the PCR reaction, there will be excess template. Please reduce the template input volume to half and check whether it works. Best of luck..
I would not say that you have a bad batch of reagents. Since you mentioned that your colleagues are also getting unspecific bands, it is likely that the cycler is not able to maintain its temperature. When was the equipment last serviced and checked?
In addition to this, it could also be nice to check that the Mg you were using earlier and the one you are using now have the same stock concentration. Excess Mg in the reaction will also produce smears.
Also, I find it strange that your stock primers are being maintained at 2uM. Does every body in your lab use 2uM stock primers? Please check around.
Totally agree with Sivadas: this looks definately like an excess of starting material. When you isolate DNA using kits, you usually get fragments between 10 and 20kb. Also with beads you cannot get rid of these bands as they will stick better to the beads than your (shorter sized) PCR products. When running a PCR, always measure your template input prior to adding it to the reaction. For genomic DNA: use approx. 50 ng per reaction.
Agree with Sivadas and Michiel... I faced the same problem in my PCR. I reduced the template in reductions of 0.5uL. Worked well for me.
High concentration template, or contaminated primers/dNTP. Since the conditions are the same from last time it cant' be that.
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