Protocol Online logo
Top : Forum Archives: : Molecular Biology

Southern Blot - The controls works but the sample not!!! (Jan/27/2005 )

Hello everyone..

Is my first time that I do this, but I am almost crazy with my problem in the southern
I am trying to do a southern to check the homologous recombination of ES cells, but before to do it with the original samples (mutated), I am trying to see if the probe work in one normal sample (wild type), both mutated and wild type, have the sequence of the probe..
Firs I tried with the first probe, but the control (which is the same probe)worked and the sample not... in that moment I thought that was the probe, so I decide 4 diffrent probes, But still with all of them is the same result the control worked but the sample not, I have tried everything, I changed the botting method (from SSC to alkaline method) and I confirm that the blotting is good because I didn't see any residual DNA in the gel after blotting. I Have tried until 1 pg of the control and as low like that worked. I have tried until 30ug of the sample concentrated in 80 ul of total volume and the quality of the DNA is ok, but nothing change the result...
I don't know what to do... I know that the procedure that I am doing is ok because the control works, but why the samples not???? Waht is wrong?

Please if somebody can help me I will be very gratefful...
Some extra information:
I am digesting with sacI, or FspI or ApaI all of them overnight incubation at 37C
I am running in 0.7% agarose gel overnight at 23 volts
I am using alkali blotting 24H capillarity blotting
I am labeling my probes with alkaline phosphatase with the kit RPN3680from amersham
I am doing the hibridization overnight incubation at 55C
I am development with chemioluminiscence

Thanks so much ....



Hi Olga
did i understand right, that your control is only the DNA of the probe, and your sample genomic DNA? if so, may be possible you are not running enough of the sample - for bacterial genomic DNA, i run 100 to 300 ng of digested genomic DNA per lane
luck, don't give up