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Preference for larger of 2 bands?

genotyping PCR size

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#1 assembler01

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Posted 30 March 2012 - 10:47 AM

I've been having trouble optimizing a new genotpying pcr reaction. I've noticed it often gives only one band instead of two for the hets (wt/flox). With this set of primers, it should give a larger band for floxed alleles and a smaller band for wild type alleles. Why would it prefer to amplify the larger piece over the smaller piece? I've checked the genotype of the parents to be sure the mating is correct. It sometimes works, but usually doesn't, in a seemingly random pattern. Sometimes it won't work one day for a particular mouse, but will work the next day or vice versa. It always works fine for just a wild type or just a flox/flox. The genotyping for our other line of mice has always been working just fine, even alongside a set of the new reactions. I would uderstand if it gave only the smaller band and then I would increase the extension time to allow for the amplification of the larger size, but this is the opposite.

Thanks.

#2 Trof

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Posted 30 March 2012 - 12:03 PM

I would make a "heterozygote" sample from validated 100% wt and flox by mixing them 50/50. That way you can test if the weirdness is in your reaction or in your samples. You can run it alongside your samples each time.

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#3 assembler01

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Posted 02 April 2012 - 12:58 PM

Mixing the DNA of the wt and f/f in the reaction gives 2 clear bands. One of the "het" mice (offspring of +/+ x f/f) still gives a single band though in the same set of reactions. It's the larger band again as well.





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