3 replies to this topic

[march]

Hi
  I want to use my DNA for real time PCR to see whether my IP dna fraction is really enriched when compared to the non-immuno precipitated reference dna.
so can i just use my dna sample for real time pcr.

[Clare]

march, on Mar 10 2009, 06:32 AM, said:

Hi
  I want to use my DNA for real time PCR to see whether my IP dna fraction is really enriched when compared to the non-immuno precipitated reference dna.
so can i just use my dna sample for real time pcr.

Are you doing chip? Yup - you can use the DNA after reverse-crosslinking and purification
Clare

[march]

Clare, on Mar 10 2009, 03:37 AM, said:

march, on Mar 10 2009, 06:32 AM, said:

Hi
  I want to use my DNA for real time PCR to see whether my IP dna fraction is really enriched when compared to the non-immuno precipitated reference dna.
so can i just use my dna sample for real time pcr.

Are you doing chip? Yup - you can use the DNA after reverse-crosslinking and purification
Clare

hi clare,
are there kits for reverse cross linking and is purification needed after ectraction with phenol-chloroform

[Clare]

hi clare,
are there kits for reverse cross linking and is purification needed after ectraction with phenol-chloroform
[/quote]


Hi again
You don't need a 'kit' for reverse cross linking, just add RNAseA (1ug) and NaCl to a final conc. of 0.3M. Incubate 67degC for a min. 5 hours (we just do overnight). Then add 60ug proteinase K at 45degC for 2 hours. We then use a QIAGEN kit to purify our DNA (the gel extraction kit, following instructions for PCR cleanup I believe). We find we get much better results doing the cleanup with a kit rather than phenol chloroform.

Hope this helps
Clare