A | B | |
Component | Volume (ul) | |
LunaScript | 1 | |
Template RNA | 5.5 | |
Total | 6.5 |
A | B | |
Temperature (°C) | Time | |
25 °C | 2 minutes | |
55 °C | 10 minutes | |
95 °C | 1 minute | |
4 | Hold |
Component | Pool 1 volumes (μl) | Pool 2 volumes (μl) | |
5X Q5 Reaction Buffer | 2.5 | 2.5 | |
10mM dNTPs | 0.25 | 0.25 | |
Q5 Hot Start DNA Polymerase | 0.125 | 0.125 | |
Primer Pool 1 or 2 (10μM) | 1.8 | 1.8 | |
Nuclease-free water | 5.4 | 5.4 | |
Total | 10 | 10 |
Step | Temperature (°C) | Time | Cycles | |
Heat Activation | 98 | 30 seconds | 1 | |
Denaturation | 98 | 15 seconds | 35 | |
Annealing | 65 | 5 minutes | ||
Hold | 4 | ∞ |
A | B | C | |
BLT(bead-linked transposomes) | 2°C to 8°C | Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting. | |
TB1(Tagmentation buffer) | -25°C to -15°C | Bring to room temperature. Vortex to mix. |
A | B | |
Component | Volume (μl) per sample | |
BLT | 1 | |
TB1 | 1 | |
Total | 2 |
Temperature (°C) | Time | |
55 | 15 minutes | |
10 | Hold |
A | B | C | |
Item | Storage | Instructions | |
TSB (Tagment stop buffer) | 15 oC to 30 oC | If precipitates are observed, heat at 37°C for 10 minutes, and then vortex until precipitates are dissolved. Use at room temperature. |
A | B | |
Temperature (°C) | Time | |
37 | 15 minutes | |
10 | Hold |
Item | Storage | Instructions | |
EPM (Enhanced PCR Mix) | -25°C to-15°C | Thaw on ice. Invert to mix, then briefly centrifuge. | |
Index Adapters Tubes/plates | -25°C to-15°C | Thaw at room temperature. [Tubes] Vortex to mix, then centrifuge briefly. [Plates] Spin briefly before use. |
A | B | |
Component | Volume (μl) per sample | |
EPM | 2 | |
NFW (Nuclease Free Water) | 2 | |
Total | 4 |
Temperature (°C) | Time | ||
68°C | 3 minutes | ||
98°C | 3 minutes | ||
98°C | 45 seconds | 8 cycles | |
62°C | 30 seconds | ||
68°C | 2 minutes | ||
68°C | 1 minute | ||
10°C | Hold |
A | B | C | |
Item | Storage | Instructions | |
SPB (Sample Purification Beads) | 2°C to 8°C | Let stand at room temperature for 30 minutes. Vortex and invert to mix. | |
RSB (Resuspension Buffer) | -25°C to-15°C | Thaw and bring to room temperature. Vortex to mix. |
Final Concentration | 20pM denatured DNA | 1% PhiX | Pre-chilledHT1 | |
12 pM | 354.6μl | 3.6μl | 240μl |