easyDB – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis
Jason D Limberis
Abstract
We designed primers with a tail sequence that forms a six-nucleotide hairpin at temperature <55oC, but not ≥55oC. These primers contain six phosphorothioate bonds starting at the complementary region to inhibit exonuclease T7 activity. The primers successfully amplified the target and, following incubation with a mixture of T7 exonuclease, DNA polymerase, and Taq DNA ligase, pseudo-circular double-stranded DNA formed.
Attachments
Steps
Prepare Buffers
| A | B |
|---|---|
| ISO buffer (2.5X) | Volume (ul) |
| 1M Tris-HCl pH 7.5 | 100 |
| 200mM MgCl2 | 50 |
| 100mM dGTP | 2 |
| 100mM dATP | 2 |
| 100mM dTTP | 2 |
| 100mM dCTP | 2 |
| 100mM DTT | 100 |
| 40% PEG 8000 | 90 |
| 50 mM NAD | 20 |
Aliquot 100μl and store at -20°C for up to two years
| A | B |
|---|---|
| easyDB Master Mix | Volume (ul) |
| 2.5X ISO buffer | 640 |
| T7 exonuclease (10 U/μl) | 0.64 |
| 2 U/μl Phusion polymerase | 20 |
| 40 U/μl Taq DNA ligase | 160 |
| H20 | 379.36 |
Aliquot 10 μl and store at -20°C
Amplicon PCR
| A | B |
|---|---|
| Component | Volume (ul) |
| 5X Reaction Buffer | 10 |
| 5X Q5 High GC Enhancer | 10 |
| 10 mM dNTPs | 1 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| DNA (5ng) | 2 |
| Q5 High-Fidelity DNA Polymerase | 1.5 |
| Nuclease-Free Water | 20.5 |
| A | B | C | D |
|---|---|---|---|
| Step | Temp (C) | Time (s) | Cycles |
| Denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 10 | 34 |
| Annealing | 62 | 10 | |
| Extension | 72 | 20 | |
| Extension | 72 | 2 | 1 |
Cycle parameters
Add 40µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on magnet
Wash 2x with 200µL freshly-prepared 70% (v/v) ethanol
Air dry for0h 0m 30s, don't allow the beads to become cracked
Resuspend in 20µL Tris-low EDTA
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on the magnet, aspirate 20µL of the eluant into a new 200ul tube
easyDB reaction
Thaw a 10ul aliquot of easyDB Master Mix on ice
Add 5ul (~150ng) DNA to the tube
Mix thoroughly by pipetting 10X
Incubate at 50°C for 60min (will be reduced, probably to 10min)
Add 20µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on magnet
Wash 2x with 200µL freshly-prepared 70% (v/v) ethanol
Air dry for0h 0m 30s, don't allow the beads to become cracked
Resuspend in 12µL Tris-low EDTA
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on the magnet, aspirate 12µL of the eluant into a new 200ul tube
Exonuclease Treatment – optional
Optional
| A | B |
|---|---|
| Component | Volume (ul) |
| H20 | 7 |
| Cutsmart | 2 |
| DNA | 10 |
| Exonuclease VIII, truncated | 0.5 |
| Exonuclease III | 0.5 |
Add 20µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on magnet
Wash 2x with 200µL freshly-prepared 70% (v/v) ethanol
Air dry for0h 0m 30s, don't allow the beads to become cracked
Resuspend in 12µL Tris-low EDTA
Mix by pipetting 10x
Incubate 0h 5m 0s at 65Room temperature
Place on the magnet, aspirate 20µL of the eluant into a new 200ul tube
