Hi all,
I ask for your help to solve a difficult problem I'm experiencing with some samples.
I'm performing ChIP-qPCR of a transcription factor. We have done this experiment many times and it has always worked with the typical variation due to changes in antibody batch.
However, the last four times I'm doing the experiment I'm getting a problem with the qPCR: some primers amplify as always and others amplify with a lot of variability between triplicates or in the case of the IP / IgGs do not amplify at all.
For your information, we are using a Roche Lightcycler 480 and as a dye we use Eva Green without ROX.
Until now, we have done the following tests:
-Try primers with old chip samples: WORK!
-Try primers with generic genomic DNA samples in different concentrations: work!
-Try to dilute our ChIP samples (1:10 to 1:1000) to see if that specific region is so overrepresented that the reaction is being inhibited (strange, as I say we have everything quite well set-up): no better amplification!
-Try primers of close regions (e.g proximal promoter --> distal promoter): in one case they amplify in other case they don't.
This problem is recurrent, the last 3 times I did the experiment happened...
Could anyone help?
Thanks in advance!!!
Raquel
Thanks for the answer.
DNA is not contaminated, it works nicely for other primer pairs. The Tm that appears is the same as before in some wells, but still, triplicates give a lot of variability.
I've already tried other primers for close regions and in one case I get amplification and in other case I don't.
The amount I am precipitating is more or less the same (as I can see in other tested regions). That's why it's so strange to get "promoter specific lack of amplification".
DNA is properly decrosslinked (as measured by agarose gel...), but for sure something out of the ordinary is happening...
Thanks for your time again!
Raquel
Hi Raquel,
Do you have the problem only with your ChIP samples, or also with your Input samples? Are you using the same cells/cell lines as before? If not, can you exclude that your amplicon doesn't contain a SNP or other genomic change that prevents it from being amplified?
Daan
Thanks for your answer Daan.
I had the problem in input and IPs. Cells were the same.
Last time I performed the experiment it worked, I still don't know what the problem was but let's hope it does not happen again.
Thanks for your time!
Raquel
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