Wagner Trout 4 (4Trout)

Setup Notes

~28 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

• Cleanup each full pool via ultra purification

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

• Mix of on campus and 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: