Feb 28, 2023
easyDB – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis
- 1University of California, San Francisco
Protocol Citation: Jason D Limberis 2023. easyDB – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j24rlo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 17, 2022
Last Modified: February 28, 2023
Protocol Integer ID: 70165
Keywords: circular, rolling circle amplification, RCA, sequencing , genotypic bedaquiline resistance testing of mycobacterium tuberculosis, genotypic bedaquiline resistance testing of mycobacterium, mycobacterium tuberculosis, genotypic bedaquiline resistance testing, mycobacterium, nucleotide hairpin at temperature, dna polymerase, nucleotide hairpin, stranded dna, circularization of rv0678, dna, taq dna ligase, easydb
Funders Acknowledgements:
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Grant ID: 1R01AI153213-01A1
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
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Materials
Rv0678 amplification primers, you can tack the tail (GGCGTCTCAAAACGCCCGTtargetedPrimerSeq) onto any primer set but remember to add the PTO modifications.
| A | B | |
| Forward primer | GGCGTCTCAAAACGCCCGT*T*T*T*C*T*GTTGGTGCTGATATTGC | |
| Reverse primer | GGCGTCTCAAAACGCCCGT*A*C*T*T*GCCTGTCGCTCTATCTTC |
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Q5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Optional
Exonuclease III (E.coli) - 5,000 unitsNew England BiolabsCatalog #M0206S
Exonuclease VIII truncatedNew England BiolabsCatalog #M0545S
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Reagents for buffers
beta-Nicotinamide adenine dinucleotide (NAD+) - 0.2 mlNew England BiolabsCatalog #B9007S
100mM dNTPs
Polyethylene Glycol 8000
Dithiothreitol
T7 Exonuclease - 5,000 unitsNew England BiolabsCatalog #M0263L
Phusion polymerase
Taq DNA Ligase - 10,000 unitsNew England BiolabsCatalog #M0208L
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 40 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on magnet
Wash 2x with 200 µL freshly-prepared 70 % (v/v) ethanol
Air dry for00:00:30 , don't allow the beads to become cracked
Resuspend in 20 µL Tris-low EDTA
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on the magnet, aspirate 20 µL of the eluant into a new 200ul tube
10m 30s
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 20 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on magnet
Wash 2x with 200 µL freshly-prepared 70 % (v/v) ethanol
Air dry for00:00:30 , don't allow the beads to become cracked
Resuspend in 12 µL Tris-low EDTA
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on the magnet, aspirate 12 µL of the eluant into a new 200ul tube
10m 30s
Exonuclease Treatment – optional
10m 30s
Optional
| A | B | |
| Component | Volume (ul) | |
| H20 | 7 | |
| Cutsmart | 2 | |
| DNA | 10 | |
| Exonuclease VIII, truncated | 0.5 | |
| Exonuclease III | 0.5 |
Add 20 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on magnet
Wash 2x with 200 µL freshly-prepared 70 % (v/v) ethanol
Air dry for00:00:30 , don't allow the beads to become cracked
Resuspend in 12 µL Tris-low EDTA
Mix by pipetting 10x
Incubate 00:05:00 at Room temperature
Place on the magnet, aspirate 20 µL of the eluant into a new 200ul tube
10m 30s