Labelling DNA for ChIP-ChIP - A couple of questions... (Apr/04/2008 )
Hi everyone! Happy Friday 
We are so close now to getting our ChIP-chip sorted that it's getting very exciting 
We just have a couple of issues with the labelling step as we have been told different things by different people.
So...after labelling DNA do you...
a) calculate how much DNA you have (we use nanodrop) and hybridise equal amounts of DNA? It seems that the latest paper from Peggy Farnham's lab does this.
or
just load whatever DNA you end up with after labelling (assuming dye incorporation is similiar)?
We are currently labelling 2ug DNA and are unsure whether we should do a or b.
Hope someone can can enlighten us!
Ok I am still confused after talking to a few people about my question...
One lab tells me to just use all the DNA after labelling as the analysis step of the array normalises the data anyway...
Another lab tells me to use equal amounts of DNA (although labelling efficiencies for the two dyes are different).
Soo....I wonder which is the best method for data analysis? Same amounts of DNA with different dye incorporations? Or evrything?
Hmmm....
HI
I am hybridizing on the basis of Picomoles/ul . after nanodrop we normalize for Picomoles/ul for both the dyes. and we are getting good results
You may be able to get yourself more confused by reading this
TGIF.http://search.vadlo.com/b/q?sn=158621799&a...eling&rel=0
..
You may be able to get yourself more confused by reading this
TGIF.http://search.vadlo.com/b/q?sn=158621799&a...eling&rel=0
..
Aye, Happy Friday! Our labelling is fine now - we are labelling 2ug of DNA and then put everything on the array. Our problem now is that the intensity of the spots isn't very high :/ But that could be due to the patient samples we use..... what a mammoth experiment!
Are you working in some microarray core facility or what?
Because as far as I know, people generally submit their DNA and let core care of everything else.
Anyways, good that your labelling started working before the weekend
[/quote]
Are you working in some microarray core facility or what?
Because as far as I know, people generally submit their DNA and let core care of everything else.
Anyways, good that your labelling started working before the weekend
[/quote]
Nope, I don't work in a core facility. I am doing the entire chip-chip on my own, even designed the array myself and am learning how to analyse my data with R/Bioconductor. I never thought I would end up doing arrays! Just shows how research can take you into wierd and wonderful directions