ChIP PCR problems - I can't get rid of the smears! - (Nov/05/2007 )

Hi everyone,

I know there have been previous posts about PCR smears but mine is slightly different.

I don't use a kit to perform my ChIP assays, I make the solutions myself and recently got the ChIP assay working very nicely. However, it seems over the last few weeks that samples which I have previosuly gotten to work produce non-specific smears or just no band when I try to perform PCR reactions using the same or different primers. I've changed everything from dNTPs, to new primers, to fresh water, to new Taq Polymerase to using filter tips and I just don't know what's wrong.

I specced my samples using a nanodrop and was getting concentrations of around 10ng/ul or higher which I thought was fairly low but they have worked in the past.

Any suggestions about what I can do?

My PCR programme is: 95oC 5 mins
95oC 30 secs
59oC 30 secs (Middle three steps x 42 cycles)
72oC 1 min
72oC 5 mins

Any suggestions would be very very appreciated!!

Thanks H

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Do you get smears with your inputs?
How about genomic DNA?
If you get smears with input then your primers or PCR solutions are gone "off".
However, on occasion (twice) I have gotten smeary PCRs with my ChIP pull downs and not my Inputs. This has something to do with the processing during the ChIP procedure (I am not sure exactly what).
Once I see the smears I throw out everything and start over.
Usually, the better the primers the less likely this will happen.

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Hi Mike,

Thanks for replying.

Sometimes I do get smears with my inputs but other times no input at all. I used genomic DNA and also got a smear. However, I used all my standard PCR reactants like water, dNTPs etc in a non-ChIP pcr and they seemed fine. I've also ordered lots of new primers. I just don't know what else to do. Do you think that it could be my ChIP samples?

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Are you dissolving your DNA is water or a buffer at pH of 8 or so? If your DNA is at too low a pH it might be autolysing. Also, is there any possibility you're getting nuclease contamination?

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Hi KPDE,

I'm resuspending my DNA in water (which is from Sigma and is supposedly nuclease free). Aside from using filter tips have you any other suggestions to how I can eliminate nuclease contamination?

Many thanks

H

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My question about nuclease contamination was a long shot since it's often not a problem with double stranded DNA (esp if you don't leave it for long periods of time at RT). Unless you are specifically crosscontaminating because you work with a nuclease quite often at your bench and are spreading it around all over everything then it's probably not the culprit. You might try dissolving your DNA in a buffer at pH 8 since it will be more stable at higher pHs. Purified water can be at a pH lower than neutral (in my experience it often is) and is not necessarily ideal for dissolving DNA.

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If you get smearing with genomic DNA (non-ChIP) then the primers are a problem.
I always work out the condition for PCR on ChIP Inputs that I know are of good quality.
Genomic DNA should also be okay.
I would suggest to get a set of primers that have been shown to work well on Inputs and
use them to verify your Inputs are of good quality.
Once you have high quality input (or genomic DNA) work out the conditions for you PCR.
Maybe you need to increase the annealing temperature or add DMSO to get more specific binding.
If you are unable to get published primers (or primers that others have used successfully) to work with
your Inputs then your ChIP procedure is probably the problem. Maybe one of the solutions (I change them often)
or maybe even the phenol/chloroform.
Good luck.




Sometimes I do get smears with my inputs but other times no input at all. I used genomic DNA and also got a smear. However, I used all my standard PCR reactants like water, dNTPs etc in a non-ChIP pcr and they seemed fine. I've also ordered lots of new primers. I just don't know what else to do. Do you think that it could be my ChIP samples?
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