RePrep 57, 58, and 59 after ligation

Setup Notes

4 plates of trout to be reprepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

• Size select for 300-366bp fragments size window via Pippin Prep

• Sending Out for Sequencing

• 5% PhiX spike

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

 PCR1:

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

*pause step

Extra PCR

Add 2.155 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Pool Using PCR Plates with Assist Plus

Organize plates by Library # above, vortex, and quick spin

Use 4GbsPoolx8

Use single channel to combine 48 ul from each well of a column into a separate labeled tube.

Pippin Prep Size Select (300-366 bp select):

Run Final Product on qPCR for check and quant

Result from qPCR check :

load =800 pM

dilute 1 nM PhiX to 500 pM

Run Final Products on Tapestation for size check