SDS precipitation after adding DNA binding buffer for column purification - (Jul/04/2012 )
I'm using Zymo columns for purifying IP'ed DNA after elution with SDS and NaHCO3. SDS precipitates and I wonder if it could affect the yield of the purification.
I've tried heating the samples after adding the binding buffer and before adding the mixture to the coluns, but I wonder if Qiagen kits would be better or if there's another way to elute mi IP from Protein A Sepharose to avoid this issue.
Add 100ul of 10% Chelex100 (in ddH2O) to your sepahrose, vortex for 10sec and boil for 20min. Cool your samples and spin down at 14,000xg for 2min. Take 75ul of the supernatant and transfer to a new tube. To the remaining pellet, add 125ul ddH2O vortex for 10sec and spin as before, take 125ul of the supernatant and add it to your previous 75ul aliquot. Discard the pellet, the supernatnat containing your IP'ed DNA is suitable for direct q-PCR, but not for array.
Instead of boiling, you can also place your chelex containing samples in a ThermoMixer set at 97 degrees with 1300rpm shaking