Make a PCR2 mastermix containing Phusion, BSA, and water in the pre-PCR hood. Aliquot 18.25 µl for each reaction into strip tubes or a 96-well plate on the ice block.
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PCR2 Mastermix (1 reaction) volume final conc.
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Phusion Flash MM (ThermoFisher F-548L) = 10.0 µl 1X
2 uM Barcoded primers* = 0.75 µl 0.075 µM
BSA 20mg/ml (NEB cat# B9000S) = 0.24 µl 0.24 mg/ml*
Nuclease-free PCR grade water = 8.01 µl
Diluted PCR1 products = 1.0µl
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Total reaction volume = 20 µl
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*Please be aware these concentrations and thermocycling conditions are only a suggested starting point. You may need to optimize to ensure the highest quality PCR2 products.