I am working on genotyping mice from ear punch samples. I am looking for some tips to troubleshoot low amplification of the PCR reaction. My bands are very faint and so it is difficult to make a clear conclusion about the genotype. I am thinking to trying diluting the PCR reaction to use template for a second round of PCR, perhaps my input DNA not good enough quality.
I am currently using homemade tissue digestion buffer with proteinase K and incubating overnight at 55C. DNA is then extracted with 100% and 70% EtOH. Any improvements to this technique would be greatly appreciated.
Julie
For genotyping earclips DNA can be prepared using a very crude/simple alkaline lysis and neutralization (HotShot). No ethanol extraction is required (see https://www.med.umich.edu/tamc/HotShotDNA.pdf). The same protocol can be used on tailclips and even liver tissue. Further, I've found a lot of the time, too much DNA will cause more issues with genotyping/PCR in general rather than too little. You could try diluting your DNA 1:10 or even 1:100 and the reaction might work better.
For our standard genotyping PCR reaction:
2ul 10X thermo pol buffer
3ul Betaine
1ul dNTP mix (10mM each)
1ul MgCl2 (25mM stock)
2ul HotShot DNA
0.15ul Forward primer (10uM stock)
0.15ul Reverse primer (10uM stock)
0.125ul Taq polymerase (we purify our own, so adjust according to your Taq)
Xul H20 (final volume 20ul)
I hope this helps!
Thanks Adam!! I will try it. I suspect there is too much DNA but PI is convinced it's too little.
Hi Julie,
We followed a similar protocol to you in my old lab. However, after digestion, we precipitated the DNA in 100% isopropanol, and then washed the pellet in 70% ethanol.
I second the HotSHOT extraction - for genotyping by PCR, this crude method is more than sufficient!
I use this method followed by PCR using 5X Phire Green Reaction Buffer and Phire Hot Start II DNA Polymerase form ThermoFisher as below:
Reagent, Volume per reaction (μL)
5X Phire Green Reaction Buffer, 2.0
Phire Hot Start II DNA Polymerase, 0.2
10 mM dNTPs, 0.2
R6/2 primer mix, 0.2
Nuclease-free H2O, 5.4
DNA template (from HotSHOT extraction), 2.0
Total, 10.0
Good luck!
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