I have attempted two southern blot procedures that ultimately failed. Two putative transformed plants, negative control plant, and positive control plasmid were studied for the blots. Extraction, digestion, gel electrophoresis, and positively charged nylon membrane transfers appeared successful. Two different techniques were used- biotin labelled probe and DIG random primer labelling.
For the first (and darker) blot, a biotin labelled probe was used and the blot was left overnight In the dark for detection. The blot looks exactly the same as the gel transferred onto it and it appears to be overcooked or have a lot of background issues occurring.
For the second attempt, DIG starter kit I was purchased along with a DIG Block and Wash set. It seems only the positive control plasmid has appeared and seems to look similar to gel as well.
Any suggestions on what problems are occurring and how to fix them are highly appreciated.
Hi Rebecca,
I would suggest watching this tutorial and explanation of southern blotting! This video might help you to figure out what went wrong. I hope this is helpful!
https://www.youtube.com/watch?v=ztEwX4uPNOE
This protocol might be helpful-
http://www.dartmouth.edu/~staphy/protocol/southern_with_dig_probe.html
It might help to run a couple of dilutions of the plasmid just to get a feel for what range of dna is detectable in this system ( dig labelling). Check also that the enzyme used does not cut the target dna making the signal weaker. You need to get some kind of positive signal. Clearly the probe is annealing but the plasmid is small so its signal is very strong because the target is a large proportion of the genome (plasmid sequence)but the amount of target in the genomic dna is very much lower so although the probe can anneal to the plasmid I would try to get a signal from the genomic dna by reducing the stringency of the wash solution post hybridisation even if this does result in higher non specific background. You can always wash again at higher stringency if you do get a signal.
It may be that the blot is working and your probable positive samples are, in fact, negative. Try a pcr of a short fragment of the probe sequence on one of the dna samples to show that it does contain the target sequence
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