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inconsistent PCR contamination (band in negative control)

inconsistent PCR contamination (band in negative control) - PCR - Polymerase Chain Reaction Forum

inconsistent PCR contamination (band in negative control) - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 06-10-2010, 07:15 PM
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Default inconsistent PCR contamination (band in negative control)



I am a beginner with PCR and working with mitochondrial primers and insects. In particular, I have created primers (several pairs) to amplify aphid DNA fragments from the COI and cytochrome b genes in the range of 150-250bp. I have had to switch primers for other reasons, and I am now inconsistently getting bands of the same size as my target DNA in my negative controls, which has been going on for several PCRs now with several different primer pairs.

I thought it was me (and still could be), but Iíve switched primers, switched kits, used other polymerases, and the band seems to appear at least every few PCRs. Iíve switched pipettes, UV radiated my pipettes, changed water, autoclaved my tubes and racks, ordered new primer stocks, but it keeps coming back. I also load my negative control first in a separate tube and set it aside before loading any of my positives. Iím running 35-40 cycles (need several cycles because the DNA is in low concentrations in my samples and partially degraded), using 1.5% agarose gels, and post-staining them. I thought this might be from carry over contamination when loading gels, but I have loaded my negative controls far away from other wells and same problem.

Iím still wondering if it is from gel loading. I didnít have any contamination for about 30 PCRs (several in another lab), and now I canít seem to get away from it. The bands are sometimes very bright and sometimes faint.

Any advice would be great because this is very frustrating and a huge waste of time. I have no problem amplifying my positive control; itís just the contamination issue.
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Old 06-10-2010, 09:18 PM
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Default Re: inconsistent PCR contamination (band in negative control)

How do you prepare the negative control for the PCR?. Your primers might be contaminated with your template DNA.

Try running a reaction with no template, and if the band appears, prepare another aliquot of your primers and do all over again. I guess this is the best hypothesis, because it's very unlikely to have the exactly same band with another template (in case you had "environmental" DNA contamination) and the same PCR program.

I asked how were you preparing the negative control, because some people add no template to the reaction, but sometimes you need to add DNA template in order to demonstrate you are not amplifying anything else than your target DNA (in example, if you want to demonstrate you have cloned something into a plasmid, you have to add the linear plasmid with no insert in it to enseure you're not amplifying the plasmid instead of your cloned fragment)
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Old 06-10-2010, 10:11 PM
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Default Re: inconsistent PCR contamination (band in negative control)

Thanks for your suggestion.
My negative control has no template and water only. I have tried your suggestion before and found bands. I replaced my primers and the contamination returned within four PCRs.
I have to run a lot of PCRs because I'm using PCR for presence/absence. I separate the pre and post PCR areas. I'm not sure what it is that I'm doing, but it keeps coming back.
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Old 06-14-2010, 12:53 PM
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Default Re: inconsistent PCR contamination (band in negative control)

If it keeps coming back after a couple of PCR, it's likely to be yourself cross-contaminating your own primer aliquots, because if it were another reason, it'd come back in the first PCR you run again.

Try adding your primers last to the PCR mix. If you're constantly cross contaminating your primers with template DNA, replace the contaminated aliquot and next time you do the mixes, add the primers at last, and see how it goes for you
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