Low blotting efficiency (southern blotting) - Low blotting efficiency (southern blotting) (Mar/31/2005 )

Hey


I have a problem with getting all of my DNA (genomic and plasmid mix) transfered from my 0.7% agarose gel (SeaKem LE) to my membrane (Nytran N membrane, Schleicher & Schuell...)

The total DNA is digested with Hind III before electroforesis, and the gel is run at 80 V for 3 hours. The gel has been depurinated with 0,25M HCl in about 10 min, before denaturation and neutralization. The blotting procedure are performed with a turbo blotting system and with 20x SSC as transfer buffer. Blotting time that has been tried out is 12 hours, 46 hours.
12 hours gives a poor transfer, while 46h gives a bit better transfer. How can I increase the transfer?

I have tried a alkaline transfer, but that doesn`t give any better result.

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hi, what you might want to try is a downward capillary transfer as described by chomczynski in analytical biochem 1992, 201, p134-39. this method is really fast and yields nearly 100% transfer efficiency after 2-3 hours of blotting (as judged by staining the membrane in methylene blue after transfer). some details: gel treatment: depurinate your gel in 0.2 M HCl until the bromphenol blue turns yellow, denature in 0.4M NaOH/1.5M NaCl until the color changes back to blue and simply assemble the blot as described in the paper; use 0.4M NaOH /1.5M NaCl as the transfer solution. after blotting for 2-3 h, dismantle the blot, soak the membrane in neutralization buffer (0.5M Tris pH7.2, 1M NaCl), dry it for a few minutes and stain in 0.02% methylene blue/0.3M NaOAc. you should see the bands of your marker and the genomic dna after a few minutes in that solution; apart from the technique itself, i'd rather change the membrane to hybond n+ from amersham. cheers.

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