DNA purification after sonication - (Aug/22/2008 )

Reversal of crosslinking is very important for looking at your fragment sizes on agarose. You will mostly get high molecular weight smears (sometimes the DNA will remain in the well) unless you reverse the crosslinks.

I do a huge volume of ChIPs every week so we have a quick way (~35min) to make input DNA for running on the gel and which is also PCR ready (as long as you don't have SDS is your sonication buffer above 0.1%). Add your chromatin (10ul or so depending on the concentration) to 100ul of 25mM Tris (pH 9.8 or above; yes I know it's out of the buffer range) with 1mM EDTA. Add 20ug of proteinase K and incubate at 55C for 15min. Then heat at 100C for 15min to reverse crosslinks (this is important for running on the gel; and yes your DNA will be fine since it's at a high pH). Then centrifuge at 4C to cool the DNA and run on a 1% agarose gel. The two most important things are to make sure that the 100C incubation is 95C or above and that the pH of the tris buffer is above 9.8 (if you make the buffer with tris base the pH, unadjusted, will usually be about 10 which is perfect).

Reversal of crosslinking is very important for looking at your fragment sizes on agarose. You will mostly get high molecular weight smears (sometimes the DNA will remain in the well) unless you reverse the crosslinks.

I do a huge volume of ChIPs every week so we have a quick way (~35min) to make input DNA for running on the gel and which is also PCR ready (as long as you don't have SDS is your sonication buffer above 0.1%). Add your chromatin (10ul or so depending on the concentration) to 100ul of 25mM Tris (pH 9.8 or above; yes I know it's out of the buffer range) with 1mM EDTA. Add 20ug of proteinase K and incubate at 55C for 15min. Then heat at 100C for 15min to reverse crosslinks (this is important for running on the gel; and yes your DNA will be fine since it's at a high pH). Then centrifuge at 4C to cool the DNA and run on a 1% agarose gel. The two most important things are to make sure that the 100C incubation is 95C or above and that the pH of the tris buffer is above 9.8 (if you make the buffer with tris base the pH, unadjusted, will usually be about 10 which is perfect).

Reversal of crosslinking is very important for looking at your fragment sizes on agarose. You will mostly get high molecular weight smears (sometimes the DNA will remain in the well) unless you reverse the crosslinks.

I do a huge volume of ChIPs every week so we have a quick way (~35min) to make input DNA for running on the gel and which is also PCR ready (as long as you don't have SDS is your sonication buffer above 0.1%). Add your chromatin (10ul or so depending on the concentration) to 100ul of 25mM Tris (pH 9.8 or above; yes I know it's out of the buffer range) with 1mM EDTA. Add 20ug of proteinase K and incubate at 55C for 15min. Then heat at 100C for 15min to reverse crosslinks (this is important for running on the gel; and yes your DNA will be fine since it's at a high pH). Then centrifuge at 4C to cool the DNA and run on a 1% agarose gel. The two most important things are to make sure that the 100C incubation is 95C or above and that the pH of the tris buffer is above 9.8 (if you make the buffer with tris base the pH, unadjusted, will usually be about 10 which is perfect).