A | B | C | |
Component | Volume | Final conc. | |
Template DNA | x µL | ||
10 µM FW/RV primer mix | 0.5 µL | 0.2 µM each | |
2X KAPA2G Robust HS ReadyMix | 12.5 µL | 1X | |
Water | 12 - x µL | ||
Total | 25 µL |
A | B | |
Primer | Sequence | |
Forward (FW) | 5'-TTTCTGTTGGTGCTGATATTGC - target-specific sequence -3' | |
Reverse (RV) | 5'-ACTTGCCTGTCGCTCTATCTTC - target-specific sequence -3' |
A | B | C | D | |
Step | Temperature | Time | Cycles | |
Initial denaturation | 95°C | 3 min | 1 | |
Denaturation | 95°C | 15 sec | 25-35 | |
Annealing | 55°C | 15 sec | ||
Extension | 72°C | 30 sec | ||
Hold | 4°C | ∞ | 1 |
A | B | |
Component | Volume | |
1st PCR products | 1.0 µL | |
BP01–12 | 0.5 µL | |
2X KAPA2G Robust HS ReadyMix | 12.5 µL | |
Water | 11 µL | |
Total | 25 µL |
A | B | C | D | |
Step | Temperature | Time | Cycles | |
Initial denaturation | 95°C | 3 min | 1 | |
Denaturation | 95°C | 15 sec | 8–10 | |
Annealing | 62°C | 15 sec | ||
Extension | 72°C | 30 sec | ||
Hold | 4°C | ∞ | 1 |
A | B | |
Component | Volume | |
2nd PCR products | 24 µL | |
AMPure XP | 12 µL |
A | B | C | |
Component | Volume | DNA | |
Sample #01 (25 ng/µL) | 1.0 µL | 25 ng | |
Sample #02 (25 ng/µL) | 1.0 µL | 25 ng | |
Sample #03 (25 ng/µL) | 1.0 µL | 25 ng | |
Sample #04 (25 ng/µL) | 1.0 µL | 25 ng | |
TN buffer | 6.0 µL | - | |
Total | 10 µL | 100 ng |
A | B | |
Component | Volume | |
Flush Tether (FLT) | 30 µL | |
Flush Buffer (FB) | 1.17 mL | |
Total | 1.2 mL |
A | B | |
Component | Volume | |
Library (from Step 29) | 11 µl | |
Water | 4.5 µl | |
Sequencing Buffer (SQB) | 34 µl | |
Loading Beads (LB) | 25.5 µl | |
Total | 75 µl |
A | B | |
Parameter | Setting | |
Flow cell type | FLO-MIN106 (R9.4.1) | |
Kit | PCR Barcoding Kit SQK-PBK004 | |
Basecalling | On | |
Basecalling configuration | Fast basecalling | |
Barcoding | On | |
Trim barcodes | On | |
Barcode both ends | Off | |
Mid-read barcode filtering | On | |
Q score filtering | 8 (default value) |
A | B | |
Component | Volume | |
Wash Mix (WMX) | 2 µL | |
Wash Diluent (DIL) | 398 µL | |
Total | 400 µL |