Feb 28, 2023

Public workspaceeasyDB – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis

DOI
dx.doi.org/10.17504/protocols.io.3byl4j24rlo5/v1
  • 1University of California, San Francisco
Jason D Limberis
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DOI: dx.doi.org/10.17504/protocols.io.3byl4j24rlo5/v1
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 16, 2022
Last Modified: February 28, 2023
Protocol Integer ID: 70165
Keywords: circular, rolling circle amplification, RCA, sequencing
Funders Acknowledgement:
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.
AB
Forward primerGGCGTCTCAAAACGCCCGT*T*T*T*C*T*GTTGGTGCTGATATTGC
Reverse primerGGCGTCTCAAAACGCCCGT*A*C*T*T*GCCTGTCGCTCTATCTTC

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ReagentQ5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880


Optional

ReagentExonuclease III (E.coli) - 5,000 unitsNew England BiolabsCatalog #M0206S
ReagentExonuclease VIII truncatedNew England BiolabsCatalog #M0545S

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Reagents for buffers
Reagentbeta-Nicotinamide adenine dinucleotide (NAD+) - 0.2 mlNew England BiolabsCatalog #B9007S
100mM dNTPs
ReagentPolyethylene Glycol 8000
Dithiothreitol
ReagentT7 Exonuclease - 5,000 unitsNew England BiolabsCatalog #M0263L
Phusion polymerase
ReagentTaq DNA Ligase - 10,000 unitsNew England BiolabsCatalog #M0208L
Prepare Buffers
Prepare Buffers


AB
ISO buffer (2.5X)Volume (ul)
1M Tris-HCl pH 7.5100
200mM MgCl250
100mM dGTP2
100mM dATP2
100mM dTTP2
100mM dCTP2
100mM DTT100
40% PEG 800090
50 mM NAD20
Aliquot 100μl and store at -20°C for up to two years
AB
easyDB Master MixVolume (ul)
2.5X ISO buffer640
T7 exonuclease (10 U/μl)0.64
2 U/μl Phusion polymerase20
40 U/μl Taq DNA ligase160
H20379.36
Aliquot 10 μl and store at -20°C
Amplicon PCR
Amplicon PCR
AB
ComponentVolume (ul)
5X Reaction Buffer10
5X Q5 High GC Enhancer10
10 mM dNTPs1
Forward primer2.5
Reverse primer2.5
DNA (5ng)2
Q5 High-Fidelity DNA Polymerase1.5
Nuclease-Free Water20.5
ABCD
StepTemp (C)Time (s)Cycles
Denaturation98301
Denaturation981034
Annealing6210
Extension7220
Extension7221
Cycle parameters

Add Amount40 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on magnet
Wash 2x with Amount200 µL freshly-prepared Concentration70 % (v/v) ethanol
Air dry forDuration00:00:30 , don't allow the beads to become cracked
Resuspend in Amount20 µL Tris-low EDTA
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on the magnet, aspirate Amount20 µL of the eluant into a new 200ul tube
10m 30s
easyDB reaction
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 Amount20 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on magnet
Wash 2x with Amount200 µL freshly-prepared Concentration70 % (v/v) ethanol
Air dry forDuration00:00:30 , don't allow the beads to become cracked
Resuspend in Amount12 µL Tris-low EDTA
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on the magnet, aspirate Amount12 µL of the eluant into a new 200ul tube
10m 30s
Exonuclease Treatment – optional
Exonuclease Treatment – optional
10m 30s
10m 30s
Optional
AB
ComponentVolume (ul)
H207
Cutsmart2
DNA10
Exonuclease VIII, truncated0.5
Exonuclease III0.5
Add Amount20 µL of resuspended AMPure XP beads
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on magnet
Wash 2x with Amount200 µL freshly-prepared Concentration70 % (v/v) ethanol
Air dry forDuration00:00:30 , don't allow the beads to become cracked
Resuspend in Amount12 µL Tris-low EDTA
Mix by pipetting 10x
Incubate Duration00:05:00 at TemperatureRoom temperature
Place on the magnet, aspirate Amount20 µL of the eluant into a new 200ul tube
10m 30s