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Neeeeed Help with my CHIP! Input value so high~


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5 replies to this topic

#1 dogest002

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Posted 28 March 2012 - 09:58 AM

Hi guys,
I'm new to this technique and I really meet a problem right here~

Here is my final Q-PCR result for my Chip experiments

Input CT: 31
IgG:33.7
IP with Ab:32.02

So there is really a pull-down like 3-fold compared to IgG

But I don't understand why the INput value is so high~

Here is how I proceed:
after sonication(around 5M cells in 500ul lysis buffer) dilute 10 times and incubate with beads to get rid off those nonspecific binding DNAs,
then took 500ul out as INPUT the rest separate to do IgG and AB pull down~~

the CT value should be at least around 24 but don't know why is 31~~

Any suggestions? 3X very very much for the help~ Really don't know how to interpret the data~ Is my CHIP works or not?

Best
Jason

#2 KPDE

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Posted 29 March 2012 - 02:33 PM

Looks like your starting material is a bit too dilute. You might try using more chromatin (dilute less) for each ChIP sample. If it's easy to get the material I would suggest testing a range of concentrations.

#3 dogest002

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Posted 30 March 2012 - 12:30 PM

THX~~ I will try it~~ But do you think my CHIP works? I mean if the INPUT is so low like this?

#4 dogest002

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Posted 30 March 2012 - 12:31 PM

Looks like your starting material is a bit too dilute. You might try using more chromatin (dilute less) for each ChIP sample. If it's easy to get the material I would suggest testing a range of concentrations.


THX~~ I will try it~~ But do you think my CHIP works? I mean if the INPUT is so low like this?

#5 KPDE

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Posted 30 March 2012 - 08:13 PM

I guess it depends on whether you trust your qPCR results at those high Cts. In my own case with the equipment I use, I don't trust Cts above 31.

#6 chabraha

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Posted 03 April 2012 - 02:09 PM

what's the reverse cross-link and elution protocol?................might be leaving some sample behind?
Treasure Chest Wizardry




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