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-   -   ChIPseq: Strange Peaks after Gel Extraction (http://seqanswers.com/forums/showthread.php?t=8346)

cmawhinney 12-10-2010 08:23 AM

ChIPseq: Strange Peaks after Gel Extraction
 
x-posted from general: i didn't realize i posted in the wrong forum until today.

Hi,

I've been doing ChIP library preps for a good while now and never before have I seen anything like this and was hoping someone maybe has?

I am seeing two peaks in my samples. One at around 200bp and one at around 278 for my single read samples and one around 228 and one around 300 for my paired end samples. It doesn't makes any sense to me since I normally take my gel slices around 250bp. It also doesn't make any sense as to why there are two separate peaks.

These samples were gel extracted, cleaned up on a qiagen column and then PCR amplified and cleaned up with SPRI beads.

The ONLY thing I did differently this time was play around with the adapter concentrations. Meaning if anything I added less adapter then I usually do. I ran 6 samples (4 SR and 2 PE) and some on different gels. All of my samples had these double peaks.

Please see the attached pictures from the bioanalyzer run. (please see this thread for the photos: http://seqanswers.com/forums/showthread.php?t=8325

Any input is GREATLY appreciated.

Chris.

DMO 12-10-2010 09:10 AM

What primer concentration are you using?

cmawhinney 12-10-2010 09:18 AM

My primer concentration varied depending on the concentration of the ChIP DNA. For example, Illumina recommends 25uM primers for 10ng of DNA. If my sample was 1ng I would dilute the primers 10 fold. This is something I routinely do.

I was concerned about my qubit readings for the ChIP samples (seemed lower then normal). If that reading was off then because I dilute the adapters and primers those would've been off as well... but not sure that would've caused these double peaks.

DMO 12-10-2010 09:23 AM

Well if the primer concentration is too low then you would start to see a tail or a second larger peak. It doesn't sound like you should be running out of primer though, unless your quantifications are off.


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