lightcycler controls and standard for CHIP - (Jul/25/2005 )

Hi everyone,

I am going to do some chippies (for HP1, hist methy and a TF) and want to quantify them using the Lightcycler. I don't have any of the regions cloned in a plasmid, so what is the best thing to use for generating the standard curve? Will you use genomic DNA from the same cell line, quantify that and make serial dilutions? if so how much (ng?) to start with?

And can someone tell what other controls are good to do and the best way to normalise the data ?

(I assume one uses the standard to quantify the input, use that to normalise ip's and then subtract the background?)

and a bonus question. would any one of you could have tried chip on satellite DNA? if so you don't freak out if you see multiple bands, right?

My brain has over-heated...

thanks in advance

Medea

-medea-

QUOTE (medea @ Jul 25 2005, 07:47 AM)

and a bonus question. would any one of you could have tried chip on satellite DNA? if so you don't freak out if you see multiple bands, right?

nope if you do see multiple bands from satelite, it is just multiples of the monomer seqeunce.

I would freak out if you are seeing smears and not specific bands!

Good luck!

Nick

-methylnick-

re: satellites

thanks a lot, that's one less worry

and about the standards, the way i think to do it is right, right?

just want to make sure before I go ahead and mess up the experiment

medea

-medea-

most Chip data I have seen in the literature and how we analyse our qPCR data is relative fold expression(amplification) against the input fraction, this does not require the generation of the standard curve.

N

-methylnick-