A | B | C | D | |
COMPONENT | 50uL REACTION | 25uL REACTION | 12.5uL REACTION | |
Nuclease-free water | 34.5 | 16.25 | 8.125 | |
5X Phusion HF or GC Buffer | 10 | 5 | 2.5 | |
10 mM dNTPs | 1 | 0.5 | 0.25 | |
10 uM Forward Primer | 1 | 0.5 | 0.25 | |
10 uM Reverse Primer | 1 | 0.5 | 0.25 | |
Polymerase | 0.5 | 0.25 | 0.125 | |
Template DNA (lysis) | 2 | 2 | 1 | |
Total amount of Master Mix | 48 | 23 | 11.5 |
A | B | C | |
STEP | TEMP | TIME | |
Initial Denaturation | 98°C | 30 seconds | |
13-16 Cycles | 98°C | 5 seconds | |
70°C | 20 seconds | ||
72°C | 15 seconds | ||
Final Extension | 72°C | 2 minutes | |
Hold | 10°C |
A | B | C | D | |
COMPONENT | 50uL REACTION | 25uL REACTION | 12.5uL REACTION | |
Nuclease-free water | 33.5 | 16.25 | 8.125 | |
5X Phusion HF or GC Buffer | 10 | 5 | 2.5 | |
10 mM dNTPs | 1 | 0.5 | 0.25 | |
Polymerase | 0.5 | 0.25 | 0.125 | |
5 uM Forward and Reverse Primer Barcodes | 4 | 2 | 1 | |
Template DNA (product of PCR I) | 1 | 1 | 0.5 | |
Total amount of Master Mix | 46 | 22 | 11 |
A | B | C | |
STEP | TEMP | TIME | |
Initial Denaturation | 98°C | 30 seconds | |
5 Cycles | 98°C | 5 seconds | |
70°C | 20 seconds | ||
72°C | 15 seconds | ||
Final Extension | 72°C | 2 minutes | |
Hold | 10°C |