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dna for real time


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3 replies to this topic

#1 march

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Posted 09 March 2009 - 10:32 PM

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.

#2 Clare

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Posted 10 March 2009 - 02:37 AM

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 :D
Clare

#3 march

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Posted 11 March 2009 - 12:22 AM

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 :wacko:
Clare



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

#4 Clare

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Posted 11 March 2009 - 03:39 AM

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 :ph34r:
Clare




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