I'm preparing Illumina TruSeq DNA libraries from MNase digested DNA, and getting weirdly sized output.
I start with MNAse-digested dsDNA that has been gel purified to contain 100-200 bp fragments, with a prominent peak at 147 bp. MNase-cleaved DNA should have 3' phosphates.
I input 1 ug of this into the TruSeq DNA kit, performing end-repair, A-tailing, and adapter ligation according to the protocol.
I run this on a gel. I'd expect peaks at 150 bp (no adapters ligated) and possibly 270 bp (adapters ligated). Instead I see very little material in the 100-400 bp range, but a smear between 500-800 bp with possible peaks near 500 and 800 bp.
I can gel purify this large stuff and amplify it according to the TruSeq PCR protocol, producing a size XL library.
Any ideas about what is going on here? Some kind of concatamerization? I was worried that the Ampure beads were size selecting against the relatively small MNAse digestion fragments, so I repeated the procedure substituting MinElute for Ampure, and got the same oversized library.
Thanks for any and all input.
I start with MNAse-digested dsDNA that has been gel purified to contain 100-200 bp fragments, with a prominent peak at 147 bp. MNase-cleaved DNA should have 3' phosphates.
I input 1 ug of this into the TruSeq DNA kit, performing end-repair, A-tailing, and adapter ligation according to the protocol.
I run this on a gel. I'd expect peaks at 150 bp (no adapters ligated) and possibly 270 bp (adapters ligated). Instead I see very little material in the 100-400 bp range, but a smear between 500-800 bp with possible peaks near 500 and 800 bp.
I can gel purify this large stuff and amplify it according to the TruSeq PCR protocol, producing a size XL library.
Any ideas about what is going on here? Some kind of concatamerization? I was worried that the Ampure beads were size selecting against the relatively small MNAse digestion fragments, so I repeated the procedure substituting MinElute for Ampure, and got the same oversized library.
Thanks for any and all input.