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Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house

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 (10 uM working)
•  Illpcr1 (10 uM working)
•  Pool of above 2 primers (5 uM working of each)

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(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 20 16 plates using the Benchsmart

Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.

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

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We are running duplicate PCRs for each template.

Add 16 ul of PCR1 MM to each well of plates

Add 4 ul of restriction/ligation products to each well

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

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