RePrep 49, 50, 51 and 53

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

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Quantify and normalize samples:

• Normalize plate reader with TE for all plates

• Quantify all plates

• Normalize first 3 to 10 ng/ul and others to 30 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 32 plates using the 8 channel

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.

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