5536 pcrs, 2 plates only get 10 columns
PCR2 Mastermix Preparation:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 5536 | 16800 |
0.45 | 10M dNTPs | 5536 | 2520 |
0.3 | Kapa HiFi HotStart DNA Pol | 5536 | 1680* |
0.5 ul | 5 uM F and R FlowCell Primers | 5536 | 2800 |
0.75 | HPLC H2O | 5536 | 4200 |
5 | Total Volume | 5536 | 28000 |
*Will need 7 tubes of Taq.
Distribute 145 ul to all wells of 2 plates. Use BenchSmart 96 well pipette to distribute 5 ul to 56 plates. Use 8 channel for the final two 10 column plates.
Use 96 channel to transfer 10ul PCR1 to appropriately labeled PCR2 redos.
qPCR Check:
qPCR gives a concentration of 12-16 nMoles/ul. Success!