PstI-MspI GBS protocol

The “protocol” that we have been using is available here as a Google doc. I use quotes there because, as you will see, there is no single protocol used by the lab to date. Without analyzed sequence data yet to compare the described protocols, the best advice I can give to you is to pick a sensible pipeline for your needs, taking into consideration the time/effort/desired output that works for you, and apply that pipeline consistently to all samples for the same project. All sets of methods described have resulted in libraries that pass QC and generate a sufficient reads during sequencing. Good luck!

PstI digest tests

Edit: I forgot to thank DanB and Kate for the generous donation of DNA. ¬†Sorry guys ūüôĀ

Edit 2: After the lab meeting some questions have been asked.

To summarize :

1.  All the enzymes work equally well.  There is a slight performance decrease if one uses the PstI-HF.  

2. ¬†I’d recommend using the PstI from Invitrogen – Invitrogen works extremely well and is the cheapest among the Psts tested ( $22 versus $75¬† ). ¬†EDIT: Brook and Kate let me know that I failed to factor in the units provided for a given dollar value. ¬†Invitrogen provides the cheapest enzyme.

For NEB – With our volume purchases we get 10000U for $71.40.

For Invitrogen – With our volume purchases we get 10000U for $63.90 {represents price negotiated with Helen, accounts manager at Invitrogen Stores}

Thanks guys for pointing that out!

This adds up considering how much we’re going through.

3. Sunflower gDNA does not digest completely in 3 hours.  It is recommended to go overnight or 18 hours with your digestion.

4. After 18 hours do not be alarmed by incomplete digestion.  This is OK according to RFLP work performed by Loren.  There should be a sufficient number of fragments for GBS libraries.

Hi all,

Here are the results from the PstI digest tests:

All enzymes tested fail to fully digest sunflower gDNA.

Enzymes from Invitrogen, Thermofisher/fermentas, NEB (non HF) and NEB HF were tested.  All performed equivalently.  I would say the NEB HF was slightly less processive after 70 min.  See attached PDF for gels and full documentation of reaction conditions.

**** 2013-Oct-08-debono-digesttest *****