Proteinase K step - (Sep/08/2011 )

I'm using the Millipore protocol. After reversing the crosslinks with 5M NaCl, I then proceed by adding 0.5M EDTA, 1M TrisHCl pH 6.5 and 10mg/ml proteinase K to my samples, then heat for 1 hour for 45 minutes. After that the protocol states that you carry on with DNA extraction via column spins or phenol/chloroform.

However, there is no mention of needing to denature the proteinase K.

Is there any need to do this?

Thanks

Dear Dave,

you MUST perform phenol/chloroform extraction . If you use the columns for extracting DNA you will not get any results because usually your DNA fragments you are generating for the ChIP are too small to be recovered with the columns.

Phenol/Chloroform extraction will capture the proteins in your sample, just be careful not to take anything from the phase in the middle, so you will not need to deactivate proteinase K.

Good luck
Nicole

I'm pretty sure using the qiagen mini-elute or PCR purification columns will be ok. I know I have used them and they seemed to work fine. You can boil your samples for ~10-20 min, assuming your buffer is pH~10, to inactivate the ProK.

I just check the Qiaquick PCR purification handbook and it says it is suitable to purify fragments 100-10,000bp. Im not saying you wont loose some of your DNA on the column or in the flow through, but it works for me.