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Problem with qPCR of ChIP input - (Jun/04/2008 )

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Have you tried running PCR on serial dilutions of your input , just to make sure that the PCR signal responds linearly with the dilutions. If there's something inhibitory to PCR in your input it will become very apparent when running dilutions.

QUOTE (chipnewbie @ Jun 17 2008, 11:44 AM)
Hi!

Sorry I've been MIA - I had commencement!

So I took your advice and took out input material without beads, but it made no difference in the PCR! I still get IP>Input!!! I am at a loss as to what to do. The protocol was working fine before, and so I've tried reverting to my old, supposedly non-optimized protocol to see if that will fix things... But I will know some time later this week after I process everything.

Basically, my automation is done using a microfluidic chip, where I load the cells into this plastic chip, and then all the lysing and everything happens on the chip. However, this means I can't sonicate the DNA, so I use the Micrococcal Nuclease digestion method instead...

Anyways, if anyone has ANY other ideas about this at all, I'd be grateful and happy to try out anything!

Thanks again for your inputs!

Angela

QUOTE (Davo @ Jun 9 2008, 06:23 PM)
If you can avoid the washing steps with your automation, avoid adding the beads with your automation. If they are agarose beads, it may be casuing PCR problems if there is any carry-over of the agarose. At the 95 degree stage of the PCR, the agarose would probably melt and cause all sorts of problems when it is cooled again to annealing temp and even at 72 it could stilll be a solid.

-KPDE-

That's a great idea. I didn't think of that! I will try that. Unfortunately the other things that were suggested didn't really fix things... I may just have to get new reagents for everything if it turns out there's contamination.



QUOTE (KPDE @ Jun 20 2008, 02:28 PM)
Have you tried running PCR on serial dilutions of your input , just to make sure that the PCR signal responds linearly with the dilutions. If there's something inhibitory to PCR in your input it will become very apparent when running dilutions.

QUOTE (chipnewbie @ Jun 17 2008, 11:44 AM)
Hi!

Sorry I've been MIA - I had commencement!

So I took your advice and took out input material without beads, but it made no difference in the PCR! I still get IP>Input!!! I am at a loss as to what to do. The protocol was working fine before, and so I've tried reverting to my old, supposedly non-optimized protocol to see if that will fix things... But I will know some time later this week after I process everything.

Basically, my automation is done using a microfluidic chip, where I load the cells into this plastic chip, and then all the lysing and everything happens on the chip. However, this means I can't sonicate the DNA, so I use the Micrococcal Nuclease digestion method instead...

Anyways, if anyone has ANY other ideas about this at all, I'd be grateful and happy to try out anything!

Thanks again for your inputs!

Angela

QUOTE (Davo @ Jun 9 2008, 06:23 PM)
If you can avoid the washing steps with your automation, avoid adding the beads with your automation. If they are agarose beads, it may be casuing PCR problems if there is any carry-over of the agarose. At the 95 degree stage of the PCR, the agarose would probably melt and cause all sorts of problems when it is cooled again to annealing temp and even at 72 it could stilll be a solid.



-chipnewbie-

I do not think you need to use a bead to do CHIP. With new advance in fast Chrompatin IP kit, you can do it in microplate well and finish it in 4 hours with no need to use a agarose bead or magnetic bead. There is a simple, fast chromatin IP kit available ! It is easy, reproducible doing reaction in microplate well. I tried it from {edit} and it worked very well.http://www.protocol-online.org/forums/index.php?showtopic=36949#

-johnd2008-
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