How do you label your amplified DNA? - If you use the Sigma WGA kit... (Nov/27/2007 )
Hi again everyone,
Yes...another question! For those of you who are using the Sigma WGA kit to amplify your DNA for chip-on-chip, how are you labeling?
1. Cy or alexa dyes? (I hear that Alexa Fluor are better but people in the lab next to me are using Cy dyes).
2. Which supplier/kit are you using?
A colleague here said he tried to label WGA-amplified ChIP material using Invitrogen's Bioprime labeling system and it didn't work. I'm not sure if that's because they are not designed to be used together or if it just failed in his hands.
Any advice would be greatly appreciated 
Clare
In my hands the Cydyes are more reliable/reproduceble between batches. But alot of people preferr the alexa dyes so I think you just have to test them for yourself ( your application).
My opinion is that a klenow RP labeling after an amplification (PCR MDA or whatever enzymatic amplification) is a bad idea. Most amplification procedures result in small (somewhat biassed) fragments that you again amplify/label with RP klenow, that will result in even smaller (and more biassed ) fragments. I would suggest to use RP klenow only with intact DNA.
What does Sigma suggest??
My opinion is that a klenow RP labeling after an amplification (PCR MDA or whatever enzymatic amplification) is a bad idea. Most amplification procedures result in small (somewhat biassed) fragments that you again amplify/label with RP klenow, that will result in even smaller (and more biassed ) fragments. I would suggest to use RP klenow only with intact DNA.
What does Sigma suggest??
Hi,
After much searching I found an article....Sigma labels with the Agilent Genomic DNA Labeling Kit which used Cy dyes and exo-klenow. However, this kit is stupidly expensive and I was going to go with Invitrogen's "Bioprime total genomic labeling system". It appears the same but uses Alexa dyes. As the minimum amount that can be labeled is 50ng...I assumed I would need to amplify first (as I'm doing ChIP on patient samples).
Clare
Hi,
I'm using the Sigma Whole Genome Amplification kit (WGA2) and label my amplified DNA's with the Invitrogen Bioprime Total kit. And it works fine in my hands.
I'm using the Sigma Whole Genome Amplification kit (WGA2) and label my amplified DNA's with the Invitrogen Bioprime Total kit. And it works fine in my hands.
Hi ChIPer,
Sweet. I'll have a go with the Bioprime kit
Clare
I'm curious about the outcome Clare, hope you will get the good results CHIPer got too,
I hope it works well too
I doubt I'll find out before xmas (waiting on samples) but will post in the new year .