I'm doing Southern Blot using Amersham Hybond N+ & ECL detection kit. I am getting a very faint specific band in mutant but not in positive control. I extended the exposure time from 1 hour to 2 hour but problem persists. Please suggest what should be done.
1. I have followed Sambrook protocol for transfer.
2. I have kept O/N hybridization at 55 degree follwowed by primary washing (55 degrees) and secondary washing at RT...steps as mentioned in the Amersham instructions
3. Detection reagent applied and waited for 5 mins
4. Then proceeded for X ray exposure
5. I extended exposure time from 1 hour to 2 hours
6. Background of blot is clear but band is very faint
7. Band got only in experimental but not getting in positive control
Please suggest what to do.
Could you please clarify: Are you getting the desired band in your positive control, or are you getting nothing? Is the size of the band in the mutant lane the size you are expecting for the positive control?
When I increased the amount of positive control DNA I got band. Ya, the size of the positive control band is same as from mutant if it there. Thanks for your response.
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