WGA2 Contamination? - (Sep/17/2008 )
Hi all,
I'm trying to amplify my ChIP samples for ChIP-on-chip using Sigma's WGA2 kit, however after amplification and column purification I'm getting the same amount of DNA in the no DNA negative control as I do in my ChIP samples. I also get a smear in my negative control when I run this on an agarose gel. In order to reduce the chance of contamination, I use fresh filter tips, aliqot each WGA2 reagent before use, pipette all reagents before opening my DNA samples (if possible) but still the smear appears. I just don't understand where this contamination is coming from, has anyone else had this problem before?
Many thanks 
Hi there,
No, we have never had that problem. How much DNA are you trying to amplify? And what size is your chromatin after sonication?
Clare
I'm trying to amplify my ChIP samples for ChIP-on-chip using Sigma's WGA2 kit, however after amplification and column purification I'm getting the same amount of DNA in the no DNA negative control as I do in my ChIP samples. I also get a smear in my negative control when I run this on an agarose gel. In order to reduce the chance of contamination, I use fresh filter tips, aliqot each WGA2 reagent before use, pipette all reagents before opening my DNA samples (if possible) but still the smear appears. I just don't understand where this contamination is coming from, has anyone else had this problem before?
Many thanks
The sonication shears the DNA to around 500-1000 bp. I resuspend all of my ChIP DNA in 10ul of water and amplify that. I'm not quite sure whether its 10ng or not as its often below the nanodrop threshold and so was giving inaccurate readings.
Cheers,
No, we have never had that problem. How much DNA are you trying to amplify? And what size is your chromatin after sonication?
Clare
I'm trying to amplify my ChIP samples for ChIP-on-chip using Sigma's WGA2 kit, however after amplification and column purification I'm getting the same amount of DNA in the no DNA negative control as I do in my ChIP samples. I also get a smear in my negative control when I run this on an agarose gel. In order to reduce the chance of contamination, I use fresh filter tips, aliqot each WGA2 reagent before use, pipette all reagents before opening my DNA samples (if possible) but still the smear appears. I just don't understand where this contamination is coming from, has anyone else had this problem before?
Many thanks
[quote name='Kitwai' date='Sep 17 2008, 03:35 PM' post='150720']
The sonication shears the DNA to around 500-1000 bp. I resuspend all of my ChIP DNA in 10ul of water and amplify that. I'm not quite sure whether its 10ng or not as its often below the nanodrop threshold and so was giving inaccurate readings.
Cheers,
How many cells are you using per IP? And are you using anti-TF or histone antibodies?
We use anti-H3 antibodies with about 6-8 million cells per IP. Typically we only have to amplify 1/2 of the IP to get enough to label (we usually get about 5ug). But with some samples we get enough DNA using 1/4 and find that using 1/2 the DNA is too much and ends up inhibiting the WGA reaction. Also, you might want to try and make fragments a little smaller. The fragmentation buffer that comes with the WGA kit shears to about 400bp, so perhaps that's the optimal size for WGA. We use fragments 200-600bp.
Hope this helps 
Clare
we have seen this with WGA2 in our hands and were told that you can get this and the DNA is non-specific and won't affect downstream applications.
We were rather worried when we saw this because you can get amplification from water?
But it was fine
Cheers Nick, I thought I was going mad or something! The only problem is that I'm following the Affymetrix protocol which requires around 7.5 ug of DNA to hybridise to an array. How is it possible to accurately quantify your amplified ChIP DNA after amplification if some of it is non specific?
Thanks again,
The simple answer is, you can't!!!
If you WGA both your input and IP fractions in the same experiment and then hybridising them to your affy array then the non-specific stuff (theoretically the same in each samples) should be the same for both and the differences you are seeing are true differences between your IP and input fractions.
It's not the best basis for your experiments but to say, know your limitations.
Nick
If you WGA both your input and IP fractions in the same experiment and then hybridising them to your affy array then the non-specific stuff (theoretically the same in each samples) should be the same for both and the differences you are seeing are true differences between your IP and input fractions.
It's not the best basis for your experiments but to say, know your limitations.
Nick
If you are in a situation where your IP fraction is below the threshold of the nanodrop how do you normalize the amount of DNA going into the WGA reaction? I would assume that you want to start with the same amount in all of your conditions (IP v input, control v experimental) since the WGA is likely to bias amplification based on the amount of starting DNA.
For our experiments we always amplify 5.5ul and 11ul of our ChIP DNA (we use the Sigma WGA kit, so 11ul if the total vol of DNA you use). Technically, the amount of DNA should double from 5.5 to 11, so if it doesn't, we use the 5.5ul sample. Same goes with our inputs - we use 15ng and 30ng per sample.
Clare