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==Preparation== *Thaw all of the reagents in Phi29 enzyme kit - except the phi29 enzyme *Thaw N6-primer (200 uM) *Prepare fresh 5M KOH (MW=56.11), and make ALS buffer: 82 uL H2O + 10 uL 1 M DTT + 8 uL 5M KOH in a 1.5-mL tube *Test the pH of ALS and NS (used the one previously made) mixture(10 uL ALS + 10 uL NS). The acceptable pH is around 7~8 ** The complete mixing by pipetting is critical in this step *UV-treatment on ALS and NS for 10 min *Set up Bio-Rad realtime PCR programing: Line 1: 30 C, 6 min Line 2: Plate read Line 3: Goto line 1 for additional 99 times Line 4: 85 C, 3 min (to kill all of the enzymes left) Line 5: 4 C, forever Line 6: End
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