A | B | |
COMPONENT | VOLUME PER ONE LIBRARY | |
DNA | 26 μl | |
(yellow) NEBNext Ultra II FS Reaction Buffer | 7 μl | |
(yellow) NEBNext Ultra II FS Enzyme Mix | 2 μl | |
Total Volume | 35 μl |
A | B | |
Duration | Temp | |
5-30 min | 37°C | |
30 min | 65°C | |
Hold | 4°C |
A | B | |
COMPONENT | VOLUME | |
FS Reaction Mixture (Step 7) | 35 μl | |
(red) NEBNext Ultra II Ligation Master Mix* | 30 μl | |
(red) NEBNext Ligation Enhancer | 1 μl | |
(red) NEBNext Adaptor for Illumina** | 2.5 μl | |
Total Volume | 68.5 μl |
A | B | |
COMPONENT | VOLUME (μl) PER REACTION | |
Adaptor Ligated DNA Fragments (Step 25) | 15 μl | |
(blue) NEBNext Ultra II Q5 Master Mix | 25 μl | |
(blue) Index Primer/i7 Primer*,** | 5 μl | |
(blue) Universal PCR Primer/i5 Primer*,** | 5 μl | |
Total Volume | 50 μl |
A | B | C | D | |
CYCLE STEP | TEMP | TIME | CYCLES | |
Initial Denaturation | 98°C | 30 seconds | 1 | |
Denaturation | 98°C | 10 seconds | 3-13* | |
Annealing/Extension | 65°C | 75 seconds | 3-13* | |
Final Extension | 65°C | 5 minutes | 1 | |
Hold | 4°C | ∞ |
A | B | |
INPUT DNA IN THE FS REACTION | # OF CYCLES REQUIRED FOR STANDARD LIBRARY PREP: YIELD ~100 ng (5–35 nM)* | |
100 ng | 3–4 | |
50 ng | 4-5 | |
10 ng | 6-7 | |
5 ng | 7-8 | |
1 ng | 8-9 | |
0.5 ng | 8-10 | |
0.1 ng | 12-13 |
A | B | |
INPUT DNA IN THE FS REACTION | # OF CYCLES REQUIRED FOR TARGET ENRICHMENT LIBRARY PREP (YIELD ~750 ng–1 μg)*: | |
100 ng | 4-5 | |
50 ng | 5-6 | |
10 ng | 8-9 | |
5 ng | 9-10 | |
1 ng | 11-12 | |
0.5 ng | 12-13 | |
0.1 ng | N/A |