A | B | C | D | E | |
Step | Regent to load | Volume (μL) | P1200M pipette mode | Incubation at room temp. | |
1 | 100% ethyl alcohol | 700 | Prime/Treat | - | |
2 | Air | 700 | Prime/Treat | - | |
3 | Room temp. Cartridge Wash Buffer 1 (Cat. No. 650000060) | 700 | Prime/Treat | 1min | |
4 | Air | 700 | Prime/Treat | - | |
5 | Room temp. Cartridge Wash Buffer 1 (Cat. No. 650000060) | 700 | Prime/Treat | 10min | |
6 | Air | 700 | Prime/Treat | - | |
7 | Room temp. Cartridge Wash Buffer 2 (Cat. No. 650000061) | 700 | Prime/Treat | <4h |
A | B | C | |
Material to load | Volume (μL) | Pipette mode | |
Air | 700 | Prime/Treat | |
Set P1200M pipette to Cell Load mode. Pipet-mix the cell suspension with a manual P1000 pipette. | |||
Cell suspension | 575 | Cell Load |
A | B | C | |
Material to load | Volume (μL) | Pipette mode | |
Air | 700 | Prime/Treat | |
Set P1200M pipette to Bead Load mode. Use a manual P1000 to gently pipet-mix beads in cold Sample Buffer (Cat. No. 650000062). Immediately load. | |||
Cell Capture Beads | 630 | Bead Load |
A | B | C | |
Material to load | Volume (μL) | Pipette modea | |
Air | 700 | Wash | |
Cold Sample Buffer (Cat. No. 650000062) | 700 | Wash | |
Air | 700 | Wash | |
Cold Sample Buffer (Cat. No. 650000062) | 700 | Wash |
A | B | C | |
Material to load | Volume (μL) | Pipette mode | |
Lysis Buffer with DTT | 550 | Lysis |
A | B | |
Component | Volume (μL) | |
RT Buffer | 40 | |
dNTP | 20 | |
RT 0.1 M DTT | 10 | |
Bead RT/PCR Enhancer | 12 | |
RNase Inhibitor | 10 | |
Reverse Transcriptase | 10 | |
Nuclease-free water | 92 | |
Total | 194 |
A | B | |
Component | Volume (μL) | |
10x Exonuclease I reaction buffer (BD Express, Cat. No. 650000071) | 20 | |
Exonuclease I (BD Express, Cat. No. 650000072) | 10 | |
Nuclease-free water | 170 |
A | B | C | |
Component | Volume (μl ) | Final Conc | |
KAPA HiFi HotStart 2x ReadyMix | 100 | 1x | |
Universal Oligo (BD Express, 650000074) | 6 | 0.3 μM | |
TSO PCR primer (10μM) | 6 | 0.3μM | |
Nuclease-free water | up to 200μl |
A | B | C | D | |
Step | Cycles | Temperature | Time | |
Hot start | 1 | 95°C | 3 min | |
Denaturation | 11 | 98°C | 20 s | |
Annealing | 62°C | 3 min | ||
Extension | 72°C | 4 min | ||
Final extension | 1 | 72°C | 2 min | |
Hold | 1 | 4°C | ∞ |
A | B | C | |
Component | Volume (μl ) | Final Conc | |
KAPA HiFi HotStart 2x ReadyMix | 500 | 1x | |
Library Forward Primer (BD Express,91-1085) | 3 | 0.3 μM | |
TSO lengthen primer (10μM) | 3 | 0.3μM | |
PCR1 product (50ng) | |||
Nuclease-free water | up to 100μl |
A | B | C | D | |
Step | Cycles | Temperature | Time | |
Hot start | 1 | 95°C | 2 min | |
Denaturation | 5 | 98°C | 20 s | |
Annealing | 62°C | 20 s | ||
Extension | 72°C | 4 min | ||
Final extension | 1 | 72°C | 2 min | |
Hold | 1 | 4°C | ∞ |