I've been trying to isolate the genome of STIV ( double stranded Circular form ~18kbp) to study the topology. I've tried using proteinase K with Tris, EDTA, SDS digest at 37 degrees Celsius overnight. Then using phenol-chloroform extraction to eliminate proteins. Then using Ethanol precipitation to concentrate extracted DNA. But most of the time the DNA, i've extracted is either nicked or relaxed circular form, which makes it hard for me to study the supercoiling form of it. Does any one have experience on extracting higher yield of supercoiling form of STIV genome?
Many thanks
Dear Chin-ya Tseng: I think you should try to raise the temp of reaction with proteinase K . An increase of the reaction temp. from 37 °C to 50–60 °C may increase the activity several times. Sometimes addition of 0.5–1% sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) and urea (4 M) also helps a lot to get the clean DNA.
I had tried raising the reaction at 50 and 55 degrees and use 0.5% of SDS within the reaction. But the best result i got is from reaction at 37 degrees. I would try with Guanidnium thiocyanate and urea. Thanks again :)
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