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DIG southern blot


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#1 cbrossin

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Posted 03 June 2011 - 08:29 AM

Please can someone give me some suggestions as to why my DIG SB is not working. Only the DIG-labeled ladder and positive control work. I have tried different DNA concentrations, less stringent hyb/washes. I know this protocol should work as it works for people in another country working on the same plant. I used the same probe and followed their protocol. the only difference is the TNA extraction (I use CTAB Doyle and Doyle), and NBT instead of CDP-star.

#2 phage434

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Posted 03 June 2011 - 09:39 AM

Start by doing serial dilutions of your probe spotted on a membrane, and detect using your NBT. This will tell you that your probe is labeled correctly and the sensitivity of your detection.

Then do a dot blot of serial dilutions of your DNA (no gel, no blotting) and hybridize with your probe to establish that the probe and DNA hybridize and can be detected. This also establishes your DNA detection sensitivity.

Only then both with the gel and blotting.

#3 cbrossin

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Posted 03 June 2011 - 09:45 AM

Start by doing serial dilutions of your probe spotted on a membrane, and detect using your NBT. This will tell you that your probe is labeled correctly and the sensitivity of your detection.

Then do a dot blot of serial dilutions of your DNA (no gel, no blotting) and hybridize with your probe to establish that the probe and DNA hybridize and can be detected. This also establishes your DNA detection sensitivity.

Only then both with the gel and blotting.






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