I'm new in southern blotting and I cannot troubleshoot my problems.
I am trying to detect the transgene copy number in my transgenic mouse. I digest 3 ug of genomic DNA using EcoR1 (unique restriction site in transgene insert). The probe is made by DIG labeling, 3.1 kb in size. EcoR1 is approximately 150 bp downstream from 3' end. I did membrane capillary transfer, prehybridization 42 degrees, hybridisation 42 degrees overnight using 2 uL/mL DIG labeled probe. High stringency wash in 68 degrees. I did the detection using CDP-Star.
In the result, I can see that the probe bound to the copy standard, even until single copy. I attached the picture of my result. My printer wasn't very good but on the live feed I can see the band in single copy standard. But, I cannot see anything on my samples (left side, 5 lanes). Also, it seems like there was high background. Since the copy standards are visible, I assume that the hybridization went well. But, why no band on my samples?
Please tell me what's wrong with my result and advice on southern hybridization. Thank you.
Btw, the white spot on the lower left of the picture is light reflection. Just to make it clear.
HI,
my 12 years experience with transgenic mouse generation and screening tells be a few posibilities:
1) Make sure you have the amount of DNA you think you have and also
you probably need to do a titration of your genomic DNA prior probing for copy numbers (if you haven't done that already).
We used 5µg of DNA most times but for some transgenes we had to go up to 10µg or even 20µg of genomic DNA to be able to detect the transgene in our mice.
2) Check the purity of your genomic DNA. Sometimes the pH or salt in buffer your DNA is diluted in or the restriction digest buffer could affect the whole reaction. Also, check your enzyme works. maybe use a fresh vial and fresh buffer.
Your transfer seems ok as your controls appeared and the labeling seems ok too.
I hope that helps
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