questions about shearing chromatin analysis
Posted 19 April 2012 - 07:11 AM
I did the chromatin shearing with Biruptor.( condition is Power "high". 10 times for 30 seconds at 30 second intervals.) The gel pic looks like a lit bit strange. I could not find the genomic DNA band on unsheared samples. Who could you give me some idea.
In the gel pic, I have 5 different concentration cell samples. The sample marked U is that unsheared sample and marked S is that sheared samples. The unsheared samples i took them before put in Biruptor and directly load on the gel without add any RNAse and incubation. I need help to continue. If i have problem with cross-link or lysis buffer.
The lysis buffer recepie is
Lysis Buffer fresh reagent [final] Tris–HCl, pH 8.0 50 mM EDTA 10 mM SDS 1% (wt/vol) protease inhibitor mix 1x PMSF 1mM Na-butyrate 20mM
Posted 23 April 2012 - 10:12 AM
Posted 03 May 2012 - 07:59 AM
I doubt my lysis step did not work and the shearing was ok.So I have repeat this lysis procedure again. This time i add the 20 gauge needle treatment and reverse cross-linking step. The result see the attachment. This time i could find the genomic DNA band in the unsheared lysis samples. So i think I could continue to next step, do you think so. Thanks:)
Posted 03 May 2012 - 08:10 AM