ChIPseq: Strange Peaks after Gel Extraction

cmawhinney · 12-09-2010, 02:04 PM

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.

Any input is GREATLY appreciated.

Chris.

pmiguel · 12-13-2010, 05:25 AM

Plus and minus amplification adaptors? In both cases it looks like the difference in size is about 70 bp. This would require your using a very small number of amplification steps after ligation, or starting with a surfeit of DNA in your ligation.

What instrument are these libraries constructed for?

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12-13-2010, 05:25 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Plus and minus amplification adaptors? In both cases it looks like the difference in size is about 70 bp. This would require your using a very small number of amplification steps after ligation, or starting with a surfeit of DNA in your ligation. What instrument are these libraries constructed for?