sonication URGENT PLEASE - (Aug/08/2006 )
hi every one,
i need to optimise my sonication conditions for CHIP. but to check the fragment sizes do i have to cross link and purify before running on a gel? can't i sonicate, centrifuge, and then run the lysate directly on the gel with out crosslinking and purifying?
thank you
you have to crosslink before sonicating because depending on the crosslinking conditions the sonication settings will be different. you have also to reverse crosslinking and purify the DNA (phenol:chloroform or columns) before running the gel: if you don´t purify the sample you won´t be able to load it on the gel; if you don´t reverse crosslink, you will lose the DNA in the organic phase (phenol:chloroform extraction). at least this is what happens in my hands.
hope that helps
i need to optimise my sonication conditions for CHIP. but to check the fragment sizes do i have to cross link and purify before running on a gel? can't i sonicate, centrifuge, and then run the lysate directly on the gel with out crosslinking and purifying?
thank you
If you would like to avoid doing the long reversal of crosslinking and phenol:chloroform extraction before running your total DNA, you can do a half hour digestion with proteinase K (20ug) and then boil for 10min in 10% chelex suspension. This is also sufficient for isolating PCR ready DNA from ChIPed samples as well.