I would love any suggestions you have for practical ways to avoid contaminating PCR reactions. What are some simple things to do? I know the basics, but I keep having sporadic contamination, so I would love some new tips. What do you do to keep your PCRs clean?
Try to use a different workspace (you can have a cabinett, though I am not sure if this is necessary) with a different sets of pipettes.
Aliquot every solution you use into small aliquots (using clean stuff of course) and throw things away regulary or when you doubt a contamination.
Depending on how much money you want or can spend, use filter tips.
Avoid aerosols which can be generated by snapping pipettes up or down.
Make sure your colleagues know and follow the rules.
I think otherwise its important to have a good and clear workflow and to follow it.
The most common cause for contamination is the water used. Make sure you have a fresh sterile stock of it aliquoted into eppendorfs. Atleast this was for me. All the best !!
I specially like this article. Is quite old (1997), but they made an extensive experiment on different conditions for PCR, maybe this could also help you.
Dirty extractions can affect the performance of your PCR, maybe an alternative method of extraction could help.
Settting up the correct conditions may avoid this problems. For example for some genotyping in plants I had contaminant amplicons at certain Tm that were avoided going up. Also, if the standard DNA concentration is not working, use 1/10 dillutions of your genomic DNA, or 1/20, 1/100.
I always use commercial water that is aliquoted in sterility conditions (reserved bottle for that purpose) to resuspend oligonucleotides when I get them (and reserve always a "virgin stock" that won't use unless an emergency).
All of this in addition to the comment of Christian.
http://www.biotechniques.com/multimedia/archive/00011/97233rr01_11727a.pdf
Hi Elizabeth. Good lab prractice is very fine, still there are other solutions to your problem unless your DNA template sample is contaminated. One solution is to use the shrimp double-strand specific DNase described in our PLoS One paper; see http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010295. You treat your (realtime) PCR reaction mixture for a couple of minutes at room remp, inactivate the enzyme by heating to 60-70 degrees C for 5-10 min, then add your template and run the reactions as usual. You can even add your template before adding the DNase provided that you have made the DNA single stranded (95 degrees C for 2 min, then on ice). The DNase is available from several sources, including Affymetrix; see http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131318#1_1 Good luck!
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