I am genotyping knockout mice with a pcr protocol that has worked very well up until this month. I use fresh mouse tails, toes, or ear snips. I lyse over night and use phenol/chloroform extraction and ethanol precipitation. I have 2 sets of primers that work very well and produce very clear bands for wild type and knockout.
I recently re-collected tissue from mice that I previously identified as Het a couple months ago. With the new DNA prep the pcr is now showing them as wild type. I've ruled out mixing up the tubes, I've kept meticulous records. The only explanation I can think of is some kind of contamination? But of what, and where? Should I throw out all my reagents and extract new DNA samples or is there another explanation?
do you get a pcr amplified product in your no dna negative control sample
ok no contamination you do not make it easy. It might be useful to re run an old dna sample to see if it is still scored as previously. If we accept that your records are accurate then the dna is not the problem unless the mice have been mixed up somehow so is it at all possible that your primer sets have been mixed up or is this a pcr product size assay? So having insulted your record keeping and your animal house work practices one last try...
Is it possible that 1 primer set ( knockout) has stopped working...is there a positive control)
Sometimes if the primers are dissolved in water they depurinate due to CO2 in the air and acid depurination and can stop working. If one set did not work all samples would look homozygous
- Badges
- Science method