Hi all,

Here is the long awaited updated GBS protocol.

PROTOCOL ->>>> GBSv2.0

There are three main changes from the previous protocol.

-After digestion and ligation, all the product is kept so more attempts can be made at the PCR.

-The PCR uses Phusion Taq, has longer extension times and one additional cycle with more primers/Taq.

-Size selection is done using AMPure beads instead of gel extraction.

These changes seem to work effectively to increase the concentration of the library. They also produce a broader library (i.e. a wider size range). We are cautiously optimistic that this will produce more sites in the end analysis. No library with this method has been sequenced yet, but two are currently with Ana, awaiting sequencing (as of Jan 29, 2012). If it turns out that the broad library is counter productive, this protocol can be modified to more aggressively size select.

Below are bioanalyzer results for two libraries from this protocol for reference.

Bioanalyzer results for Kristin ragweed GBS using protocol v2

Bioanalyzer results for GregO sunflower GBS using protocol v2