Hi everyone,
I'm looking for an alternative to sonication or benzonase for lysing DNA in Western Blot samples.
Briefly, I am performing a subcellular fractionation on 3 Mio cells with a hypotonic lysis to first lyse the plasma membrane. Then I lyse the nuclear membrane in 20ul (50 mm Tris(pH7.6), 150mM Nacl, 2 mM EDTA, 1% NP40, 1% Trition X100, 1% SDS) and add 6 ul SDS Page sample buffer (NuPage LDS Sample Buffer with DTT). I boil the sample for 10 min at 90 C before sonicating anywhere between 5 and 30 minutes but wasn't able to get rid of the gloopiness of the sample. Benzonase is obviously going to have a very tough going in the harsh lysis buffer that I'm using. I've tried decreasing the SDS concentration to 0.5 or 0.1% but that does not efficiently lyse my nuclei. With the current protocol I seem to very neatly separate cytosolic and nuclear fractions but I just can't get rid of the DNA which results in uneven loading and my gels running weirdly.
My questions are:
- Does anyone have any suggestions of what could be wrong with the protocol?
- Could boiling before vs after the sonication make a difference? I usually did it after sonication, just before loading the gel which seemed to work a bit better but a colleague suggested that before sonication might be more effective.
- Is there any other method to efficiently lyse DNA in Western Blot samples?
Incubate at 37 degrees for 1 h with DNase in the presence MgCl2 after sonication . DNase in the presence of MgCl2 will degrade all the DNA contaminants. I hope it helps.
Perhaps precipitation of nucleic acids by Poly (ethyleneimine).
Article Precipitation of Nucleic Acids with Poly (ethyleneimine)
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