shoulder peaks in TruSeq libraries

[hawaii454-0]

Hello,
I've been following the TruSeq DNA sample preparation method to make library preps from 3ug of starting DNA. I am aiming for an insert size of around 600bp. The DNA is sheared using a Covaris instrument and a band is cut out of a 1.2% long agarose gel. After 15 cycles of PCR amplification with longer extension time, the library is cleaned up (with ampure XP beads) and run on an Agilent high sensitivity chip (see picture). We have been getting shoulder peaks of between 300 to 500bp beside the larger main library peak which can affect the final analyses of the results after sequencing.

Has anyone else come across these shoulder peaks and/or been successful in removing them/creating libraries without them?

Thanks for your help,
Anna

[csquared]

When are you doing the size selection step? After shearing or after ligation?

[HESmith]

Overloading the gel or running at high current can cause poor size resolution. At this point, you can repeat the size selection of your amplified library to eliminate the shoulder.

[hawaii454-0]

Csquared, the size selection (i.e. cutting out of the gel) takes place after ligation.

HESmith, 20uL of the ligated sample is run with 5uL gel dye in one well at 90V. Is this too much? Would you suggest repeating size selection after PCR and seeing a shoulder peak? On a gel?

Thanks for your replies,
Anna

[HESmith]

The electrophoresis conditions - mass of DNA (not volume) and current (more relevant that voltage) - depend upon the size of the gel. Search the internet for suggested parameters.

And yes, you can repeat the size selection on a gel after PCR.