Genotyping by southern blot - (Nov/11/2005 )
Hi everybody.
We've tryied in our lab to produce a transgenic mouse and it all worked well up until I tryed to genotype the first litters. I've tryed PCR using primers for genomic DNA and for the supposedly inserted cDNA fragment and results were not consistent so I've moved to Southern methods.
In the southern, after performing restriction with HpaII and hybridization using 700bp probe corresponding to exonic sequence (exons 6-11 out of 12) I can see the insert clearly, but:
1. I never see the genomic DNA. (could it be that my 700bp probe is not binding the average 100-200bp exons?)
2. Using WT liver DNA I get the same band as cDNA gives... maybe RNA? but that (if still present) should migrate differently...
Any ideas why these things happen?
Thanks.
You can calculate, based on the CCGG sequence in cDNA and genomic DNA, and predict the pattern.
If you are concerning about RNA, you may add some RnaseA after HpaII digestion and incubate for a while
I have the restriction map both for cDNA and genome, so I know what band size to expect... 1.4kb for cDNA and 4.2kb for genomic (exon5-exon11). It's just that I don't see the genomic one in any of the samples while I get to see the cDNA one in the liver of a wild-type!... I've repeated the southern with tail DNA of a wild-type and the 1.4 band was gone. 
We've tryied in our lab to produce a transgenic mouse and it all worked well up until I tryed to genotype the first litters. I've tryed PCR using primers for genomic DNA and for the supposedly inserted cDNA fragment and results were not consistent so I've moved to Southern methods.
In the southern, after performing restriction with HpaII and hybridization using 700bp probe corresponding to exonic sequence (exons 6-11 out of 12) I can see the insert clearly, but:
1. I never see the genomic DNA. (could it be that my 700bp probe is not binding the average 100-200bp exons?)
2. Using WT liver DNA I get the same band as cDNA gives... maybe RNA? but that (if still present) should migrate differently...
Any ideas why these things happen?
Thanks.
Regarding PCR. You should have optimised the primers while microinjections and FO generation were raised. This is done by spiking mouse genomic DNA (negative control if mouse genomic DNA minus spiked DNA) with the transgenic cassette to determine primers are specific for the transgene and secondly to determine if your PCR method is sensitive enough to detect the transgene at a single copy level.
Regarding Southern why do you not digest part of the transgenic cassette and use this for the probe as it should have 100% homology with integrated DNA.