So this is a problem that has been inconsistent over the past year but has really become more prominent over the past few weeks - for practically every PCR I've run in order to genotype the mice we use for various genes, I will see faint product bands in the water control. Normally, I would contribute this to contamination, but instead of just seeing the same bands across all samples as you normally would if there's contamination, my samples will behave as 'normal', where either one or two bands will be present if the mouse being genotyped is heterozygous or homozygous for the gene of interest, or no band will be present at all if the mouse doesn't have the gene. If contamination is the issue, this shouldn't be the case, as I use the same water for the master mix and get varying results between the samples and the water control. My only other conclusions might be splash-over from other wells, although I tend to keep at least 1 well of space between my samples and the water control, or the formation of primer dimers, although those bands should be below 100bp, not at 200 or 300bp. Here is my typical method for preparing a PCR, in case anyone asks:
1. Prepare master mix - I do this separate from the DNA, and for the most recent PCR I replaced everything except for the primer aliquots I made a while ago
2. Add MM to pcr tubes - still separate from DNA, I follow the protocol I have for how much each sample should get
3. Add DNA to pcr tubes - I use new tips for each sample obviously, and know I'm not accidentally going into the wrong tubes and adding the wrong samples
4. Run the PCR
5. Add Loading Dye to PCR tubes
6. Load gel and run
Any thoughts? While the samples still show results that I would expect from a PCR done correctly, I don't trust them completely if the water control is acting like this, and obviously I need mice with specific genotypes for my experiments to work properly. Thanks for your help!
The best thing I can suggest is to redesign your primers and in doing so procure a new batch of primers
Also, have you tried suing another set of primers just to completely eliminate this possibility
I take your point about primer dimers although occasionally you can get primer concatamers that are bigger
Have you tried buying in molecular grade water ? I suppose it isn't impossible that a side reaction occurring in water may not occur if the specific reaction associated with your sample is outcompeting what ever is happening with exogenous DNA in your water
Use pre purchased molecular grade water; try alternative primers and perhaps redesign your primers if you find changing the primers eliminates the contaminant band
If this is an on going issue you could always purify and sequence the band to establish its exact identity but that is taking you off on a tangent so change water and primers first
did you have this contaminant band for different primer pairs that target different genes ?
If yes, do the contaminant band have always the expected size ?
Laurence is right,.. to test his explanation (primer competition with exogenous DNA), you can try a PCR reaction with a non-mouse DNA sample (human DNA or other)... just for fun :)
You can also check the primer specificity in silico by BLAST analysis.
in addition to all the above suggestions, I suggest you to rule out the presence of PCR inhibitors in your gDNA preps. Inhibitors might be blocking the PCR in sample tubes, whereas your water control gives band. This you can do with a well established primer for mouse house keeping gene. Before switching to different primer for genotyping, I suggest you to check the specificity of the current primers by primer blast.
best wishes,
SD
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