I have to choose what type of blotter to buy: capillary or semi-dry electroblotter (vacuum is unavailable for me) and do not have experience with Southern blotting. I read that electroblotting is inefficient for large fragments (>500kb), but capillary is slow and messy. Nevertheless there are some old reports as this one that used electroblotting but with large volumes of low-salt buffer and custom made aparatus (as opposed to semi-dry).
Ishihara H, Shikita M.Electroblotting of double-stranded DNA for hybridization experiments: DNA transfer is complete within 10 minutes after pulsed-field gel electrophoresis. Anal Biochem. 1990 Feb 1;184(2):207-12.
Can you please share some thoughts about it please ?
I agree with Artur. Capillary blotting always worked perfectly for me. The secret was the fragmentation. Once the DNA was fragmented, the blotting was quantitative. Since the fragmenting was a chemical reaction, DNA of any starting size was reduced to the perfect size for capillary blotting. We also used 0.5 M NaOH for the blotting medium, which made the setup a bit faster, but you don't want to splash a bunch of NaOH around the lab, so a little more care is required. Nonetheless, we always stained the gels after blotting (after neutralization of the gel) and always got complete transfer of the DNA. We never invested in the electroblotting stuff because the capillary system worked so well and was really cheap.
The high molecular weight DNA needs to fragmented prior to both capillary and electroblotting. This is a key precedure. In our hands treatment with 0.25 M hydrochloric acid for 30 mins works well. The gel is then neutralised wit 0.4-0.5 M NaOH as David mentioned. For electroblotting NaOH needs to be neutralised and gel equlibrated with the transfer buffer. For the capillary blotting this not needed and you can directly use 0.4M NaoH as a blotting buffer.
Together, I see no adavantage of electroblotting in PFGE and vote for a cheaper conventional capillary altenative.
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