ddRAD library sequencing

Reagents and Ordering

• EcoR1 (20,000 units/ml)
• MseI (10,000 units/ml)
• T4 DNA ligase buffer (400,000 units/ml)
• DNA polymerase (BioRad iProof or KAPA HiFi)
• BSA (1 mg/ml)-We have this in 20 mg/ul
• 1 M NaCl-We have this at 5M
• DMSO
• EcoR1 adaptors (We need 1 uM MIDed in plates)
• Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
• IIllpcr2 (2.5 uM working)
• Illpcr1 (2.5 uM working)

Original SOP

Normalize templates

Use plate reader to quantify templates

Normalize to between 20 ng/ul and 150 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 22 plates using the Benchsmart

Add 6 ul template to each plate with 8 channel. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour.

Adaptor Ligation

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

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Cover, vortex, and spin plates. Incubate at 16C for 6 hours.

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Add 4 ul of restriction/ligation products to each well

Extra PCR?