I was wondering if I can get some ‘hands on experience’ help with my Southern Blot experiments. I have been failing in getting anything other than a high background blot image (see picture) at the end and I cannot identify where the problem might be.
My 11 samples are digested gDNAs from transgenic mosquitoes (phenotype and PCR positive) and the probes (700-500 bp – P32 radiolabeled) target the two markers, hybridized separately – dsRed and GFP. I have been using Zeta-Probe nylon membranes and the transferring is successful. I follow closely the protocol suggested by the manufacturer and use SDS as a blocking reagent. Given that it is a multi-step procedure with lots of tweaks, I am looking for ideas on how to improve the stringency of the protocol to be able to see some bands at the end.
Thanks in advance!
I do not think that the primary problem is high background.That can be dealt with later. You need to get a hybridisation signal first and then clean things up. I can see no specific signal so I think that the problem lies either with the probes not annealing or that you are washing too stringently and washing off your signal.The probe can only bind if it is single stranded and the binding sequence exists in the sample.Check your probe dissociation conditions. I would try a very low stringency probe mix (6x ssc perhaps) and hybridise overnight. Wash with 2x ssc a couple of times and develop with intensifying screens at -80c for 24 hours then develop the film.Keep the filter wet and if there is any sign of darker specific signal over the high background then re wash at a higher stringency to try to improve the signal. If you have a guaranteed positive control add that to your sample set at the gel separation stage....diluted unlabelled ds probe perhaps added into a spare lane halfway through electrophoresis to make sure it stays on the gel or run on sample free gels while you sort out hybridisation conditions to spare valuable sample usage. When you have a positive signal troubleshooting is much easier. At some stage you may even have to try annealing and washing at a lower temperature to ensure probe binding. What are your annealing salt and temp and, wash temp,salt concentration
Hi Paul Rutland,
First, thank you for the valued suggestions!
The probes target the fluorescent markers and the DNA is from fluorescent individuals, so the sequence exists in the samples for sure – we just need to confirm the number of insertions. But I can do tests and/or include the unlabeled probe in the run next time, good idea!
I followed the protocol previously used with success in the lab, but I was not sure of the freedom of alterations I could make to it. Conditions:
- Pre-hybridization / Hybridization: 0.25 M sodium phosphate 7% SDS - 65◦C ON
- Washes: (1) 2x SSC 0.1% SDS - 65◦C 15 min / (2) 0.1x SSC 0.1% SDS - 65◦C 30 min / (3) 0.1x SSC 0.1% SDS - RT 15 min – the signal in the membrane (Geiger counter) is still pretty high after this washes that is why I figured the low stringency was the main problem.
Do you think is worth it to try this suggested low stringency (no SDS) protocol in the stripped membrane? I would appreciate any other insight you have on this. Thanks again!
I would reprobe the membrane but only wash to 2xssc at 65c then expose the filter. If you have a strong signal then wash at 65 at 0.5x ssc and develop the filter again. only if you still have a strong signal should you go to more stringent washes.
If you have a cold probe then you could dot blot serial dilutions onto nylon and hybridise and develop to test what level of target you can detect and at what level of stringency of the wash ( you could run it on a gel but it is not necessary.Dot blotting is quicker.
When you hybridise keep the volume low (concentration of probe as high as possible) and hybridise overnight.
remember it is the salt not the sds that determines stringency. Keep the sds as it keeps the blot cleaner with its detergent properties
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