I am exploring the use of 2D gel DNA electrophoresis for analyzing plasmid DNA replication in vitro. I have found a number of articles that utilize this technique for this purpose, but they all conclude the experiment with a Southern Blot, rather than simple staining to visualize the DNA. Most of the probes are simply full length, labelled copies of the plasmid, so it doesn't appear that the blotting is being used for specificity. I wonder if it is just for sensitivity, but I cannot find out if this is the reason, or just how much more sensitive the Southern Blot is. Anybody have a clue?
You can expect to see 0.1picogrammes of target dna using Southern blotting but this is for single copy sequences and if measuring multicopy targets the signal will be stronger. PCR has the ability to amplify 1000 fold in 10cycles so even single cell single copy dna can be amplified with PCR and quantification of PCR is very much better than with Southerns and about 3 days quicker. Southerns are also specific to the sequence probed while staining of bands gives no information about the sequence of the dna stained. You can see 1ng with a small well comb and not run very far in a gel but with normal equipment about 10ng is more likely to be the limit if all of the dna is in a single band which is unlikely if the dna is a plasmid
Hello Devin Camenares ,
Southern blot can locate your selective DNA fragment and labelling of probe using DIGoxi may amplify the signal several folds. However, simple staining with ethidium bromide is sensitive for more than 1ng per band concentration. This is all about sensitivity of techniques.
If you want to know further, you can extend the query.
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