Strange higher band in Bioanalyzer after size selection

[JNissen]

Hi all,
I just got the data back from 7 samples Ive run on the Bioanalyser (see attached file). All samples have been prepared with the illumina Paired-End kit and I size selected the libraries around 350pb (+/- 25bp). In more or less all the samples I see an additional band around 500-550pb.

Basically, I have no idea what this band is since the only step after the size selection is gel extraction with the qiagen kit.

Have any of you ever seen something similar? I was wondering whether it could be somekind of dimers that have been forming?

Any ideas about what it could be, how to test it and how to get rid of it will be greatly appreciated.

Thanks,

Jesper

[Gege]

Hi,
I think there may be some problems when you doing your Gel cutting. Maybe your gel cutting is not very sharp and straight.

From my view, I do not think it is the dimer. The peak around 150kb should be the primer or dimer or something.

I usually do Gel extraction again after PCR reaction.

Hope these can work for you.

[JHU-ChIPmaniac]

There was some discussion about this on another thread. The thought was that it may be due to overamplification of the adaptor ligated fragments. I use the Invitrogen E-Gel size select system to isolate libraries both before and after PCR amplfication. I also saw a very light higher molecular weight band on the gel ( as well as lower molecular weight primer dimers and adaptor complexes) but after analysis on a bioanalzer, all that was there was a tight band around 250 bp that represented my library. I'm waiting to get back my sequence.

[aperera]

This is not due to the size selection and it is not primer-dimer. We see this as well and think it has something to do with how the DNA is migrating. One thing we have noticed is that you need to take in to account the additional peak. Else, your cluster numbers will be too high.

[pzumbo]

this protocol is the epitome of unstandardized trash.

[Scotch]

Just to throw it out there for comment, but the higher peak may represent single stranded PCR product, which does not run correctly on the bioanalyzer. It occurs when there is too much DNA added to the final PCR reaction, and then there is not enough primers in the solution for the last 72C 5 minute extension. We saw it to a small extent with Agilent sureselect, but now see it a lot more with Nimblegen, probably because the Nimblegen hybridization is more efficient at pulling down DNA fragments.

[colossus]

There was some discussion about this on another thread. The thought was that it may be due to overamplification of the adaptor ligated fragments. I use the Invitrogen E-Gel size select system to isolate libraries both before and after PCR amplfication. I also saw a very light higher molecular weight band on the gel ( as well as lower molecular weight primer dimers and adaptor complexes) but after analysis on a bioanalzer, all that was there was a tight band around 250 bp that represented my library. I'm waiting to get back my sequence.

What is your results? I also see a 250 bp band in my library.