Is it necessary to measure the amount of DNA before PCR? - (Nov/01/2007 )

Really new in this field I just start doing CHIP recently. An experienced collegue said he never measure DNA concentration by picogreen before PCR, even different amount of DNA is in it, we have input and IgG should finally normalize the data by ^^Ct. sure we domeasure the input because of so much of DNA in it. I did follow what he said and the system seems working.
I just wondered that how necessary to measure how much DNA you get before you run PCR? The amount of DNA should be quite low right?

-cathy-

For normal PCR, we usually quantify DNA before adding it to the mixture.

I would guess one should know the amount of anything that one would use in experiments even if its not a big factor.

-scolix-

QUOTE (cathy @ Nov 1 2007, 09:12 AM)
Really new in this field I just start doing CHIP recently. An experienced collegue said he never measure DNA concentration by picogreen before PCR, even different amount of DNA is in it, we have input and IgG should finally normalize the data by ^^Ct. sure we domeasure the input because of so much of DNA in it. I did follow what he said and the system seems working.
I just wondered that how necessary to measure how much DNA you get before you run PCR? The amount of DNA should be quite low right?

I've never measured the amount of DNA prior to running real time. We just normalize to input (or normalize the input chromatin before running ChIP). In my case the signals are always within the linear range of my primers so there's no need to adjust before running PCR (except for the input samples which I always dilute 20 fold or so before running PCR).

-KPDE-

QUOTE (KPDE @ Nov 1 2007, 10:38 AM)
QUOTE (cathy @ Nov 1 2007, 09:12 AM)
Really new in this field I just start doing CHIP recently. An experienced collegue said he never measure DNA concentration by picogreen before PCR, even different amount of DNA is in it, we have input and IgG should finally normalize the data by ^^Ct. sure we domeasure the input because of so much of DNA in it. I did follow what he said and the system seems working.
I just wondered that how necessary to measure how much DNA you get before you run PCR? The amount of DNA should be quite low right?

I've never measured the amount of DNA prior to running real time. We just normalize to input (or normalize the input chromatin before running ChIP). In my case the signals are always within the linear range of my primers so there's no need to adjust before running PCR (except for the input samples which I always dilute 20 fold or so before running PCR).