Genetics as it applies to evolution, molecular biology, and medical aspects.
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I have sent purified plasmid DNA to mouse transgenic core to develop transgenic mice with that specific DNA piece.
Out of about 45 mice that I genotyped, only about 3 mice gave me the right bands (so I have 3 founders), indicating the presence of the foreign DNA in the mouse. I got few pups from these founders that have the foreign DNA as well. However, few months later, all my genotyping results on new pups from my founders are turning out to be negative (Dont notice any bands). I was confused and re-tailed my founders and genotyped them again. Surprisingly, I dont notice the bands in my founders that I had observed previously. I really doubt that the results I saw on my founders were plain contamination (false positive results). (Note: Not all 45 mice were genotyped at once, may be about 10 mice five different times. Also, when ever I had genotyped, I had a positive control that showed positive results and negative control that showed no bands. So No contamination with my controls). I'm really confused. did any one face the same issues that I did? Also, is there a possibility that the DNA in my 3 founders got integrated in the telomere region, so as the mice got older, the foreign DNA got disintegrated. Any suggestions/answers are welcome. Thanks
Obviously I dont know.
Am I correct to believe you are genotyping by PCR? If so, the obvious answer is that your positives were false (contamination). THis is very likely if you use this gene a lot and have PCR'd before, as its likely that your pipettes are contaminated as is your PCR machine. Even if not, are you sure you did not carry over contaminants when setting up the PCR (e.g. did you add your DNA to teh tubes last, always change tips, and did you use aerosol resistant tips? ).
Presumably you still have the original DNA - this is important. Now it could be that when you went back to your founder DNA it simply failed the PCR - did you optimise the PCR, and do you have a positve control for the PCR (e.g. actin?).
Although I may be wrong on this next point, my suspicion from what you tell me is that your PCR failed but that your founders are genuine- the big question though is whether your founders have transgenic gonads - it is possible that they are mosaic - and the transgene went into the tails but not the gametes. But as your PCR is failing even though it used to work, (PCR can be temperamental...) get that working, confirm your original finding, and run PCR reactions for the offspring along with the founders everytime because depending on your gene and primers and the purity that you want to take your DNA to (phenol chloroformed EtOh pptd well diluted DNA is best), you don't want to miss the day when the PCR Gods are smiling...Good luck.
Also, from another thread post, some of these tips will be relevant:
There's loads of things you can do to optimise PCR.
From my recollection, in order of preference, if you still need help on this:
0. Do a control reaction to test all the variables (taq, dNTP's especially, to ensure the PCR worked) - (usually someone can offer you actin primers and template at known molarity).
1. Mg concentration is key - alter this, use the temperature that gave you the best result.
2. You are using cDNA - does this mean a crude RT prep? You could do a DNA clean up to remove any proteins (a 50:50 Phenol/chloroform say 100-500ul , vortex, spin 10mins, pipette of the aqueous phase to a fresh tube. dispose of phenol carefully according to local rules (evap in a chemicals fumehood is best). Ethanol precipitate the DNA using standard protocols in Mol Biol - as your target is small, use some carrier DNA to ensure you dont lose the precipate - red-pellet precipation carriers are on the market (try SigmaAldrich).
3. Use less DNA- you will probably get better results, and save your precuious DNA.
4. Use thin wall tubes for PCR - and cut your cycle hold times (means you can get faster sample to result times - great if you have insomnia anyway/ you can shorter breaks).
5. eliminate the final step of 72oC - it is rarely needed, and if you are cloning it can cause damage to the phosphate ends and your cDNA cloning might not work.
6. reduce the initial heating step time as this can damage you enzyme.
7. Avoid too much enzyme- always use a master mix of enzyme which is well vortexed to mix- otherwise glycerol in the reaction can be variable across your samples and too high. Always keep your reactions on ice. Sometimes batches of enzyme vary and have variable shelf lives...
8. Try hot-start PCR if thats not working well.
9. Consider the possibility that your larger bands are alternative spliced variants and clone and sequence them too. But beware that PCR often creates bizarre unnatural hybrid molecules which are entirely artefact.
If non of this helps, either your technique is wrong, or cut your losses and redesign your primers. I think that covers A LOT OF it!
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