Genotyping PCR - (Feb/06/2008 )
Hello,
Since 1 month now I´ve been trying to genotyp a new knockout mouse in our lab (made in a different lab). The other lab uses Real Time PCR to genotyp the animals but we don´t have the machine. I tried different primers, but the wildtyp reaction does not work at all. I should have a specific band at 528bp, which I usually get, but I also get bands at 350, 200 and 50 (primer dimers) of equal strength.
I´m using Sigma Red Taq (but also tried others) and tried different Primer/DNA Concentrations, Gradient PCR, everything without losing those other bands.
Any help would be very appreciated,
thank you
Yvonne
Since 1 month now I´ve been trying to genotyp a new knockout mouse in our lab (made in a different lab). The other lab uses Real Time PCR to genotyp the animals but we don´t have the machine. I tried different primers, but the wildtyp reaction does not work at all. I should have a specific band at 528bp, which I usually get, but I also get bands at 350, 200 and 50 (primer dimers) of equal strength.
I´m using Sigma Red Taq (but also tried others) and tried different Primer/DNA Concentrations, Gradient PCR, everything without losing those other bands.
Any help would be very appreciated,
thank you
Yvonne
Is it necessary that you get rid of those extra bands? If you sequence your 528 bp band and it gives you what you want, then perhaps you can ignore the non-specific products. I guess it would depend on what your expectations are in the knockout mouse - if these extra bands would interfere with your interpretation of the results.
For some of the genotyping I do I also have some faint non-specific bands. However, as smu2 already pointed out, so long as you know that your band at 528 bp is specific this should be okay.
Out of curiosity, have you tried ordering different primers (essentially the same as what you have already, but plus or minus a few on each end to change the Tms?) This could increase the specificity of your reaction. Twice now I've received genotyping protocols from other labs and ended up having to slightly modify the primers from the sequence they gave me because when I checked them myself, the primers were either not specific enough, or had Tms that were much further apart than I'd like.
Ginger
Thank you all for the suggestions.
I´m quite happy with the results but my boss wants one specific band or he does not accept the result.
I´ve tried different primers with different length and annealing temperatures. I´m thinking it might be our PCR machine. Today I´ll try a PCR machine from another lab. Hopefully I´ll have better results, I´m getting tired of always doing genotyping without results:)
Thanks again
Yvonne