i need a proper protocol to make a good 12.5% poly acrylamid gel for nucleotide (50-36 bp) separation.
Hello, for such short oligos, I would recommend even higher concentration of PA gel. I think 15 % PA give you much better resolution than 12.5 % PA. Also run ELFO under denaturation conditions to achieve separation only according to oligo length (secondary structures, which alters its mobility often occur). In other respects I would recommend protocol from Peter Onuh.
Best regards
Martin
Weigh 12.5 grams of polyacrylamide crystals and add enough buffered water to make a 100 grams of solution. For details, consult the protocol in Tom Maniatis' Gene Cloning Manual, which everybody uses. Better yet, use pre-cast gels available commercially.
@ Vicente Mendoza Reyes
adding only water instead of buffer is not good idea I think. Also the polyacrylamide gel requires TEMED and APS to polymerize, so it is not so simple at all.
Carrying out the highlighted procedures above here deserves measuring accurately 12.5 g of Polyacrylamide crystals and carefully add appropriate measurements of polymerization agents such as TEMED and APS. Doing this deserves devoting 100% attention to end up in generating polyacrylamide gel for DNA extraction, separation or purification.
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