Faint Band - PCR reaction - (Oct/29/2007 )
I am genotyping transgenic mice via tail gDNA .
I perform a 2-step PCR reaction. First, I amplify the WT allele. Second, I amplify the Neo-cassette.
If I get no WT band, but a Neo band, the genotype is Homozygous for the transgene
If I get only a WT band, naturally, there is no transgene
If I get both a WT and Neo band, then it is heterzygous...
My question is:
For some samples, I get a Neo band, but I also get a very very very faint WT band. This would suggest heterzygosity, but let me stress this one more time... The WT band is very very very faint...
Would you still consider this a heterzygote, or a homozygote?
Also, the Faint band is the same size as the WT band, so it is not an un-specific band, but very faint.... although other heterzygous samples the ratios of WT to Neo bands are equal...
Do you have a relible marker to confirm you specificity?
If not, I'd try to do the PCR for alleles separatly. Mix with DNA, PCR mix and only WT primers and then the same reaction and the same DNA but only neo primers - it would give you more clear band.
work with care, this faint band may be indication of contamination of the PCR reaction (from contaminating WT DNA). Has the WT PCR been optimised? Is the WT band in heterozygotes strong?