Southern Blot Question Re: Copy Number, Other Problem - (Mar/26/2007 )

I have 3 questions. The first may seem silly, but I've been unable to find the answer elsewhere.
1) I am working with a diploid organism. When I Blot, will I see the number of genes that are across both sets of chromosome or just as if it were a haploid organism. That is to say, if a plant has 3 genes for toxin production would I see 3 bands or 6?
2) I am having a hard time getting my blot to stay moist, so that I can later strip it, while the film is exposing. I am doing this at room temperature, is there a better technique?
3) Does anyone have any experience using short (~100 bp) probes for Southern Blot? Any ideas on a good hybridization and Wash temp and Wash SSC concentration?

Thank you very much

1. I would say 3 bands. Yes, you do have 6 alleles, but only 3 genes (two copies of each gene per genome). Every two alleles of the same gene are exactly the same, and will be cut within the same restriction places (ignoring the mutations) detected with the same probe. For example you have genes A, B and C from mother and A1, B1 and C1 from father. A = A1, the same length, the same sequence, the same band .
2. I don't know . I don't strip (my blots).
3. Use standard protocol and don't go over 65 °C when washing (1. 2X SSC RT, 2. 0,5X SSC 63-65 °C; 3. optional 0,1X SSC 63-65 °C). Hybridization temperature can be as low as 42 °C, even without formamide, works for me at 45 °C. So you let your probe anneal with every possible sequence with wich has at least some homology, and then wash away mismatched sequences. Try with dot-blot first to check your temperatures and protocols. I've done 5 dot-blots for optimization before the real thing. M.

1. I would say 3 bands. Yes, you do have 6 alleles, but only 3 genes (two copies of each gene per genome). Every two alleles of the same gene are exactly the same, and will be cut within the same restriction places (ignoring the mutations) detected with the same probe. For example you have genes A, B and C from mother and A1, B1 and C1 from father. A = A1, the same length, the same sequence, the same band .
2. I don't know . I don't strip (my blots).
3. Use standard protocol and don't go over 65 °C when washing (1. 2X SSC RT, 2. 0,5X SSC 63-65 °C; 3. optional 0,1X SSC 63-65 °C). Hybridization temperature can be as low as 42 °C, even without formamide, works for me at 45 °C. So you let your probe anneal with every possible sequence with wich has at least some homology, and then wash away mismatched sequences. Try with dot-blot first to check your temperatures and protocols. I've done 5 dot-blots for optimization before the real thing. M.

I take 1, 2, 5 ug od total DNA per drop, add 1/2 V 20X SSC to sample, boil 10', chill on ice 5', and put a drop (or more drops, depends on how concentrated your DNA is, I wouldn't go over 1ug/ul) on my membrane.

For example, if you have 1ug/ul DNA solution, and want to make 2 ug drop, take 2 ul of DNA and 1 ul of 20X SSC and mix.

Then dry, UV, prehyb, hyb, detection, all as in protocol. M.