I have been using chIP for the analysis of recruitment of a protein to a promoter in a human cell line. Lately I have been having some trouble in the process.
After reversing the cross links, during ethanol precipitation of DNA, salt is precipitated along with DNA, which in turn is interfering with the PCR.
I use 1% SDS and 0.1 M NaHCO3 for elution of proteins, incubation at 65C for 4 hrs with 5 M NaCl for reversing the cross links, 1 hr incubation with 0.5 M EDTA, Tris-HCl (pH 6.5) and proteinase K, followed by recovery of DNA using phenol chloroform method.
In the last step of phenol chloroform extraction, I add double the volume of ethanol, 1/5th volume of 3M NaOAc and use LPA for precipitation and incubate overnight at -20C for the DNA precipitation. This is where salt is precipitated along with DNA.
Can anyone suggest what might be causing the precipitation of salt along with DNA?
Thanks a lot.
Hi,
I do precipitate ChIP-DNA with only 1/10 Vol 3 M NaOAc and 4 µl Glycogen (Fermentas 20 µg / µl) followed by vortexing. Then adding 3 times volume pure ethanol followed by 1 h centrifugation at 4 °C with 20,000 rcf. Pellets were washed in 500 µl 80 % EtOH keeping the pellet intact and spun again at 20,000 rcf for 5 min at 4 °C. 80% EtOH was removed completely with a pipette avoiding the pellet.
I never had salt in my samples at the end. The 70-80 % EtOH washing step takes care of this. The first precipitate can have salt, especially if you use 1/5 volume NaOAc.
Hope I could help you with this.
Cheers
Stephan
I would not put my DNA at -20 overnight. That might be causing salt to precipitate along with DNA.
Good luck!
Hi,
In addition to the comments above, you could use PCR purification columns. You resuspend the decrosslinked DNA in 5 volumes of binding buffer, load everything on the column and elute with H2O. Procedure is quick and works very well in my experience.
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