Hey,
I am a newbie who has been trying to build ChIP libraries using the TruSeq ChIP Sample preparation kit.
After doing protocol optimization we decided to use MinElute columns for pre-PCR purification instead of using Ampure beads XP. After the PCR, we did a Ampure XP clean-up to remove adapter dimers with the recommended ratio of 1:1.
For the Gel size selection, we are using the E-Gel Size-Select Gels to select fragments between 250-300bp of size.
However, now the libraries that we prepared using the Truseq protocol show a shoulder before the peak and the peak is of a much higher weight than the expected one.
What may be the reason for these shoulders? Could it be Y-shaped adapters? I don't think so, since after PCR they should just appear in a 120bp peak.
Is it possible to eliminate them even if one does gel size selection? Or is it just a normal artifact caused by a not so tight gel size selection made using E-gel?
I used the search function and couldn't find a satisfactory answer.
I am a newbie who has been trying to build ChIP libraries using the TruSeq ChIP Sample preparation kit.
After doing protocol optimization we decided to use MinElute columns for pre-PCR purification instead of using Ampure beads XP. After the PCR, we did a Ampure XP clean-up to remove adapter dimers with the recommended ratio of 1:1.
For the Gel size selection, we are using the E-Gel Size-Select Gels to select fragments between 250-300bp of size.
However, now the libraries that we prepared using the Truseq protocol show a shoulder before the peak and the peak is of a much higher weight than the expected one.
What may be the reason for these shoulders? Could it be Y-shaped adapters? I don't think so, since after PCR they should just appear in a 120bp peak.
Is it possible to eliminate them even if one does gel size selection? Or is it just a normal artifact caused by a not so tight gel size selection made using E-gel?
I used the search function and couldn't find a satisfactory answer.