Hello guys,
I have a serious problem with genotyping of IR flox mice.
First I did according the Jackson Protocol: https://www.jax.org/Protocol?stockNumber=006955&protocolID=16534
It should make 2 bands of WT=105 and Mut:145 bp but my results are different.
I can see bands of 105 and 200 and many of samples just show a smear instead of bands.
I changed the DNA extraction from homemade to REDExtract N Amp from sigma but no improvement.
The PCR kit was KAPA mouse genotyping and after that the red-extract.
I tried to change the annealing temperature but it did not improve the results.
I attached a picture of the Gel. No 1 is Jackson IR fl/fl and others are some of our mice.
I appreciate any help to solve this problem.
Best wishes,
Masoud
Hi, maybe you can add,
a. 1% betaine (0.3 to 0.5 micro liter) and
b. 10％ DMSO (1.5 ~ 2.5 micro liter) in the PCR mix and adjust the amount of water accordingly.
You didn't mention the concentration of the agarose gel you have used, but you can try to increase the concentration of the gel at the same time too (say, 2 or 2.5％). And, use fresh gel running buffer.
Hope it will help. Let me know the outcome.
Regards,
Shafiq
Thank you Md Shafiquzzaman
You are amazing!
I tried your suggestion and added 10% DMSO to the PCR mixture. And also in another group with the same DNA samples, I heated the primers for 3 min at 95C and then put them on ice.
I ran both methods together on a 3% gel and you can see the heated primers are good but DMSO (with non-heated primers) is even cleaner.
I attached a picture.
What do you think about the results?
Thank you so much for your great help.
Best regards.
Masoud
Hi,
I am really glad that it worked, the bands are very clean.
Cheers!
Best Regards.
Shafiq
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