Hello all,

I am trying to genotype some KOs mice we have made. We have deleted 2 regions of a chromosome, single deletions, and some double deletions. These regions are near each other, and there may be AT-rich (though I do not how to identify AT-rich areas).

because of the size of the deletions, my WT primers have to give me a band of 2 kb and 4 kb.

When using WT mice as a control, there are few problems amplifying these regions. however, sometimes, in heterozygous mice, I only get the KO band. A bit more info:

delA ==> WT=1.5 kb KO=750 bp for one allele and 300 bp for another allele

delb ==> WT= 3.5kb KO=2.7kb for one allele and 3kb for another allele

I have confirmed that my primers are good, and I think I have found the right annealing and extension temps. I have tried multiple conditions and the differences are little. I get my DNA by isolating it from the QIAGEN Blood and Tissue kit. We use the Gottaq (M5123 from Promega).

PCR program:

1) 95C - 30 sec to 2 mins

2) 95C - 30 sec to 1 min

3) 62C - 15 sec to 20 sec

4) 72C - 2 mins or 4 mins (depending on WT band size)

repeat steps 2 to 4 35 to 40 times

5) 72C - 5 to 10 mins

6)4C - 00(infinite)

I do not understand why in some samples I get a good band and in others a do not, and this repeats no matter how many times I test the same sample.

I have made smaller primers along the chromosome region (chromosome walking) and I get good results when extending smaller regions.

Please let me know how I can optimize my PCR genotyping for these conditions. I am about to pull all my hair.

Thanks,

Alessandro