Removal of long fragmented DNA

[Adiza]

We are using the QSONICA Q800R machine for a while, in order to have fragmented gDNA at the size of 250-300bp. After sonication we tried Thermo Scientific PCR cleanup kit, and then applied the NEBNext DNA library prep kit for Illumina. After size selection of adaptor – ligated DNA, there are still unwanted large fragments.

Do you have any ideas to remove this unwanted lenghts?

Thanks in advance,
Adi

[luc]

Hi Adiza, please search for "Ampure XP bead upper cut" protocols.
An alternative is gel based size selection; manually or with a Pippen Prep system.

[Adiza]

Hi luc

are you sure that the "upper cut" protocol fits to gDNA sonicated samples before turning it into libraries?
I've tried the E-Gel method (if that was your intention) though it's too wasteful in the step of sonicated samples...

[nucacidhunter]

250-300 bp is a very narrow cut and requires Pippin or similar automatic size selection instrument. Size selection with bead would result in broader size range.

Large fragments do not affect sequencing output and results unless for a specific purpose you want tighter size range.

If you have done size selection after adapter ligation and run it on TapeStation or Bioanalyzer you will get wrong size and possibly double peak. You should check the size after PCR purification.

[mspringer]

Hi Adiza, please search for "Ampure XP bead upper cut" protocols.

This is what we use as well. It's pretty good at removing higher length fragments.