Problems with Southern hybridization and restriction digest of genomic DNA - (Mar/07/2006 )
I tried to do Southern blotting on genomic DNA of green algae. We extracted the DNA by a CTAB methode, then digested 10 µg of gDNA with EcoRI and we got a fine smear. We blotted the gel and hybridized it with an actin probe, because we want an idea of the copy number of the actin gene in green algae. We got detection but it was a smear at the top.
What would be the problem:
1. Not completely digested: due to impurities in the DNA extract. I read that sometimes spermidine is added to enhance digesting
2. Something wrong with hybridization?
3. Something else
Al advice is more then welcome,
I would say it's incomplete digestion.
10 ug does sould like a lot of DNA for a blot. Try using 2-5 ug.
How many units of enzyme do you use and what is your incubation time.
EcoR I is cheap but is not the best enzyme in the world as it has star activity, I've none overnight digestion with plasmids and not got good results, when I've repeated the digestion over a couple of hours, I've good results.
Join the club of the unwilling Southerns! I tried many over the last months but most of them didn't give signal, only the Dig-marker lights up.
I am sure that for me the problem is the purity of the genomicDNA. I'm working with plant gDNA, Medicago and Sesbania. For Medicago the CTAB-protocol is working well but my problem is the Sesbania DNA. I can prepare a good quantity of gDNA, I digest it ON with different enzymes (including EcoRI) and I have a nice smear on my agarose-gel, blot it ON, hybridize it ON with a digprobe but I can not get signal. I tried several plant-gDNA-protocols, also a brown-algae one but until now without succes. I even tried a CsCl-prep! but I didn't have enough gDNA and it is a bit of a dirty protocol with loads of EtBr so I did't try it a second time yet.
According to me you have the same problem. If you have a nice smear on your agarose-gel the digestion of the DNA is OK but something is preventing it from hybridizing with the probe. We always do ON-digestions for Southerns and that is not the problem.
For plants it is described that you have to use young leafs, older leaf contain polyphenols and other substances that are difficult to remove from your gDNA. I don't know how to harvest "young algae..."