Reagent | Volume | |
10 mM dNTPs | 1 μL | |
Oligo library | ||
Plasmid template | ||
Q5 Enh buffer | 10 μL | |
Q5 polymerase* | 2 μL | |
Q5 Rxn buffer | 10 μL | |
Water | To 50 μL* |
Temperature | Time | |
98 °C | hot start | |
98 °C | 4 min | |
Annealing temperature* | 20 s | |
72 °C | Elongation time* | |
12 °C | hold |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1 μL | |
Purified Step1 product | * | |
10 μM Universal F1 | 2.5 μL | |
10 μM Block specific R1 | 2.5 μL | |
Q5 polymerase | 0.5 μL | |
Water | To 50 μL |
Temperature | Time | |
98 °C | hot start | |
98 °C | 3 min | |
98 °C | 8 s | |
Annealing temperature | 20 s | |
72 °C | Elongation time | |
Repeat 3-5 for 34 more cycles | ||
72 °C | 5 min | |
12 °C | hold |
Reagent | Volume | |
Type2S enzyme* | 2 μL | |
Purified Step2 product | 1.2 μg | |
Reaction buffer | 5 μL | |
Water | To 50 μL |
Temperature | Time | |
Reaction temperature (Lid: 60 °C) | 50 min | |
12 °C | hold |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1 μL | |
Purified Step3 product | ||
Plasmid template | ||
Q5 polymerase* | 2 μL | |
Water | To 50 μL |
Temperature | Time | |
98 °C | hot start | |
98 °C | 5 min | |
72 °C | 5 s | |
66 °C | 20 s | |
72 °C | Elongation time* | |
12 °C | hold |
Reagent | Volume | |
DpnI | 0.5 μL | |
MspJI | 0.5 μL | |
CutSmart | 5 μL | |
Enzyme activator | 1 μL | |
Purified Step4 product | 500 ng | |
Water | To 50 μL |
Temperature | Time | |
37 °C (Lid: 60 °C) | 1 hr | |
12 °C | Hold |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1.5 μL | |
Purified Step5 product | 100 ng | |
10 μM F2 | 2.5 μL | |
10 μM R2 | 2.5 μL | |
Q5 polymerase | 1 μL | |
Water | To 50 μL |
Temperature | Time | |
98 °C | hot start | |
98 °C | 5 min | |
98 °C | 6 s | |
Annealing temperature | 20 s | |
72 °C | Elongation time | |
Repeat 3-5 for 34 more cycles | ||
72 °C | 5 min | |
12 °C | Hold |
XbaI | 1.5 μL | |
BamHI-HF | 1.5 μL | |
Plasmid template | 3 μg | |
CutSmart | 5 μL | |
Water | To 50 μL |
37 °C (Lid: 60 °C) | 40 min | |
12 °C | Forever |
NEBuilderMaster Mix | 20 μL | |
Backbone | 210 ng | |
Purified Step6 product | 140 ng | |
Water | To 30 μL |
50 °C (Lid: 60 °C) | 60 min | |
12 °C | Forever |
Reagent | Volume | |
Electrocompetent cells | 40 μL | |
Assembly reaction | 4 μL | |
Water | 160 μL |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1 μL | |
Plasmid | ~300 ng | |
10 μM F primer | 2.5 μL | |
10 μM R primer | 2.5 μL | |
Q5 polymerase | 0.5 μL | |
Water | To 50 μL |
Temperature | Time | |
98 ºC | hot start | |
98 °C | 3 min | |
98 °C | 6 s | |
70 °C | 15 s | |
72 °C | 5 s | |
Repeat 3-5 for 32 more cycles | ||
72 °C | 5 min | |
12 °C | hold |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1 μL | |
Plasmid | ~50 ng | |
10 μM F primer | 2.5 μL | |
10 μM R primer | 2.5 μL | |
Q5 polymerase | 0.5 μL | |
Water | To 50 μL |
Temperature | Time | |
98 °C | hot start | |
98 °C | 3 min | |
98 °C | 6 s | |
Annealing temperature | 15 s | |
72 °C | 7 s | |
Repeat 3-5 for 33 more cycles | ||
72 °C | 5 min | |
12 °C | Hold |
Annealing temperature | Blocks | |
61 °C | 1, 4, 6, 7 | |
64 °C | 5, 9, 10 | |
66 °C | 2, 3, 8 |
Reagent | Volume | |
Q5 Rxn buffer | 10 μL | |
Q5 Enh buffer | 10 μL | |
10 mM dNTPs | 1 μL | |
Mixed purified products | 100 ng | |
10 μM NGS-PCR3-F | 2.5 μL | |
10 μM NGS-PCR3-R | 2.5 μL | |
Q5 polymerase | 1 μL | |
Water | To 50 μL |
Temperature | B | |
98 ºC | hot start | |
98 ºC | 3 min | |
98 ºC | 6 s | |
72 ºC | 15 s | |
72 ªC | 8 s | |
Repeat 3-5 for 19 more cycles | ||
72 °C | 5 min | |
12 ºC | Forever |
Primer name | Sequence | |
PCR1-F | GATCGTCACTTGGTACCGGTTCTAGA | |
PCR1-R (FKRP) | TGGCACTTTTCGGGGGATCCTC | |
PCR1-R (LARGE1) | TGGCACTTTTCGGGGGATCCCT |
Reagent | Catalog | Volume/weight | |
Q5 Reaction Buffer | NEB, B9027SVIAL | 10 µL | |
Q5 High GC Enhancer | NEB, B9028AVIAL | 10 µL | |
10 mM dNTPs | NEB, N0447 | 1 µL | |
Q5 High-Fidelity DNA Polymerase | NEB, M0491SVIAL | 1 µL | |
10µM PCR1-F | 2.5 µL | ||
10µM PCR1-R | 2.5 µL | ||
gDNA | 0.3-1 µg | ||
Nuclease-Free Water | To 50 µL |
Step | Temperature | Time | |
Step1 | 98 °C | Hot start | |
Step2 | 98 °C | 3 mins | |
Step3 | 98 °C | 8 s | |
Step4 | 68 °C | 20 s | |
Step5 | 72 °C | 45 s | |
Step 3-5, 35 cycles | |||
Step6 | 72 °C | 5 mins | |
Step7 | 12 °C | hold |
Reagent | Catalog | Volume/Weight | |
Q5 Reaction Buffer | NEB, B9027SVIAL | 10 µL | |
Q5 High GC Enhancer | NEB, B9028AVIAL | 10 µL | |
10 mM dNTPs | NEB, N0447 | 1 µL | |
Q5 High-Fidelity DNA Polymerase | NEB, M0491SVIAL | 1 µL | |
10µM PCR2-F | 2.5 µL | ||
10µM PCR2-R | 2.5 µL | ||
gDNA | 0.2-0.5 µg | ||
Nuclease-Free Water | To 50 µL |
Step | Temperature | Time | |
Step1 | 98 °C | Hot start | |
Step2 | 98 °C | 3 mins | |
Step3 | 98 °C | 6 s | |
Step4 | Annealing temperature | 15 s | |
Step5 | 72 °C | 7 s | |
Step 3-5, 25 cycles | |||
Step6 | 72 °C | 5 mins | |
Step7 | 12 °C | hold |
Temperature | Primers | |
61 °C | LARGE1-blk1, LARGE1-blk4, LARGE1-blk6, LARGE1-blk7 | |
64 °C | FKRP-blk1, LARGE1-blk5, LARGE1-blk9, LARGE1-blk10 | |
66 °C | LARGE1-blk2, LARGE1-blk3, LARGE1-blk8 | |
68 °C | FKRP-blk6 | |
72 °C | FKRP-blk2, FKRP-blk3, FKRP-blk4, FKRP-blk5 |
Reagent | Catalog | Time | |
Q5 Reaction Buffer | NEB, B9027SVIAL | 10 µL | |
Q5 High GC Enhancer | NEB, B9028AVIAL | 10 µL | |
10 mM dNTPs | NEB, N0447 | 1 µL | |
Q5 High-Fidelity DNA Polymerase | NEB, M0491SVIAL | 1 µL | |
10µM PCR3-F | 2.5 µL | ||
10µM PCR3-R | 2.5 µL | ||
Mixed sample | 23 µL |
Step | Temperature | Time | |
Step1 | 98 °C | Hot start | |
Step2 | 98 °C | 3 mins | |
Step3 | 98 °C | 6 s | |
Step4 | 72 °C | 15 s | |
Step5 | 72 °C | 8 s | |
Step 3-5, 25 cycles | |||
Step6 | 72 °C | 5 mins | |
Step7 | 12 °C | Infinite |