A | B | C | D | E | F | G | |
Step | Reagents | Conc. | Unit | Manufacturer | Kit/full description | Product number | |
End repair 1 | Water | Fisher chemicals | Water for HPLC 1L | 10367171 | |||
End repair 1 | NEB Buffer 2 | 10 | X | New England Biolabs | B7002S | ||
End repair 1 | dNTPs | 25 | mM | Thermo Fisher | R1121 | ||
End repair 1 | BSA | 20 | mg/ml | Thermo Fisher | B14 | ||
End repair 1 | ATP | 10 | mM | New England Biolabs | P0756S | ||
End repair 1 | T4 PNK | 10 | U/µl | New England Biolabs | M0201L | ||
End repair 1 | USER enzyme | 1 | U/µl | New England Biolabs | NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4) | E7335S, E7500S, E7710S, E7730S | |
End repair 2 | T4 Polymerase | 10 | U/µl | New England Biolabs | M0201L | ||
Adapter Ligation | Quick Ligation Buffer | 5.0 | X | New England Biolabs | NEBNext Quick Ligation Module | E6056L | |
Adapter Ligation | Adapter Mix (2.5 µM) | 2.5 | µM | *Custom made from Eurofins adapters | n/a | ||
Adapter Ligation | Quick T4 Ligase | 1.0 | U | New England Biolabs | NEBNext Quick Ligation Module | E6056L | |
Fill in | Water | Fisher chemicals | Water for HPLC 1L | 10367171 | |||
Fill in | Thermopol Buffer 10X | 10 | X | New England Biolabs | Bst DNA Polymerase, Large Fragment | M0275 | |
Fill in | dNTP 25mM | 25 | mM | Thermo Fisher | R1121 | ||
Fill in | Bst DNA polymerase | 8 | U/µl | New England Biolabs | Bst DNA Polymerase, Large Fragment | M0275 | |
PCR | Water | Fisher chemicals | Water for HPLC 1L | 10367171 | |||
PCR | 10x PCR buffer | 10 | X | Eurogentec | HGS Taq Diamond set | TAQ-I011-1000+ | |
PCR | MgCl2 | 25 | mM | Eurogentec | HGS Taq Diamond set | TAQ-I011-1000+ | |
PCR | BSA 20 mg/ml | 20 | mg/ml | Thermo Fisher | B14 | ||
PCR | dNTPs (10 mM) | 40 | mM | Thermo Fisher | R0191 | ||
PCR | HGS Taq Diamond | 1 | U | Eurogentec | HGS Taq Diamond set | TAQ-I011-1000+ | |
PCR | Dual Index Primers Set 1 | 10 | µM | New England Biolabs | NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) | E7600S | |
PCR | Dual Index Primers Set 2 | 10 | µM | New England Biolabs | NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2) | E7780S | |
Purification | PB Buffer | Qiagen | MinElute PCR Purification Kit | 19066 | |||
Purification | PE Buffer | Qiagen | MinElute PCR Purification Kit | 19065 | |||
Purification | EB Buffer | Qiagen | MinElute PCR Purification Kit | 28006 |
A | B | |
Number | Equipment and consumables | |
1 | 0.2 ml tube rack | |
2 | 1.5 ml tube rack | |
1 | 1.5 ml cool block | |
1 | 0.2 ml cool block | |
10 µl filter tips | ||
20 µl filter tips | ||
100 µl filter tips | ||
200 µl filter tips | ||
1000 µl filter tips | ||
[# of samples]×5 (tubes) | 0.2 µl PCR strips (8 tubes) | |
[# of samples]×2+5 | 1.5 ml tubes | |
1 | 5 ml tube | |
[# of samples]×2 | MinElute columns | |
1 | 50 ml Falcon (waste) |
A | B | |
Numbers | Equipment and consumables | |
1 | 0.2 ml tube rack | |
2 | 1.5 ml tube rack | |
1 | 1.5 ml cool block | |
1 | 0.2 ml cool block | |
10 µl filter tips | ||
20 µl filter tips | ||
100 µl filter tips | ||
200 µl filter tips | ||
1000 µl filter tips | ||
[# of samples]×5 (tubes) | 0.2 µl PCR strips (8 tubes) | |
[# of samples]×2+5 | 1.5 ml tubes | |
1 | 5 ml tube | |
[# of samples]×2 | MinElute columns | |
1 | 50 ml Falcon (waste) |
A | B | C | D | E | F | |
REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE | |
Water [already added] | 25.85 | |||||
NEB Buffer 2 | 10 | 1.0 | X | 7.50 | vortex | |
dNTPs | 25 | 0.30 | mM | 0.90 | vortex | |
BSA | 20 | 0.20 | mg/ml | 0.75 | vortex | |
ATP | 10 | 1.00 | mM | 7.50 | vortex | |
T4 PNK | 10 | 0.40 | U/µl | 3.00 | on ice | |
USER enzyme | 1 | 0.06 | U/µl | 4.50 | on ice | |
Master Mix total | 50.00 | |||||
Template DNA or water | 25.00 | |||||
REACTION TOTAL | 75.00 |
A | B | C | D | E | F | |
REAGENT | STOCK | FINAL | UNIT | 1× VOL (µl) | NOTE | |
T4 DNA Polymerase | 3 | 0.038 | U/µl | 3.00 | on ice | |
REACTION TOTAL |