Help on library, restriction enzyme fragmentation

[Martin Mikkelsen]

Dear Everyone,

At our lab we are doing a sequencing project sequencing a short gene (1500 kb) in humans. The length of the amplicon does not really fit any of the fragmentations tools available in our lab, so we decided to cut the DNA using restrictions enzymes instead. However, when building our library using the standard Rapid Library protocol (NEB or Roche kits) all our DNA seems to disappear somewhere between the end repair and the ligation. The restrictions enzymes creates a mix of blunt end and overlapping end.

We are running out of ideas....
- Should we just try to ligate the adaptors on without any end repair?
- Should we leave out any of the enzymes?

Any thoughts on the above or help would be very much appreciated.

Martin

[flxlex]

Have you considered PacBio sequencing instead? No need to fragment the 1500 bp product, high quality reads (circular consensus) and 1500 bp should now be possible.