Oct 07, 2023

Public workspaceBead Beating in Custom Buffer Followed by XP Bead Cleanup (NGS Workflow) 

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbz7rvx1/v1
  • 1University of California, San Francisco;
  • 2Stellenbosch University, Cape Town
  • Sacramento Public Health Laboratory
Alina Nalyvayko
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzbz7rvx1/v1
Protocol Citation: Jason D Limberis, Alina Nalyvayko, Janré Steyn, Jennifer Williams, Melanie Grobbelaar, Robin Mark Warren, john.metcalfe 2023. Bead Beating in Custom Buffer Followed by XP Bead Cleanup (NGS Workflow) . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbz7rvx1/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: May 06, 2023
Last Modified: October 07, 2023
Protocol Integer ID: 81498
Keywords: NGS, targeted sequencing, sputum, MGIT, M.tuberuculisis, DNA extraction
Funders Acknowledgements:
NIAID
Grant ID: R01AI131939
Abstract
DNA extraction method for Mycobacterium tuberculosis from various sample types as described in: "Insights into Mycobacterium tuberculosis DNA extraction for targeted deep sequencing using the Deeplex Myc-TB assay: Lessons for improved drug resistance diagnosis."
Materials

Equipment
FastPrep-24 Classic bead beating grinder and lysis system
NAME
Bead beater
TYPE
MPBio
BRAND
116004500
SKU
Reagent Screw cap micro tube 1.5 ml PCR Performance Tested Low DNA-bindingSarstedtCatalog # 72.703.700
ReagentAgencourt AmPure XP beadsContributed by usersCatalog #A63880
Reagent0.1 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079101z

Safety warnings
Work done with Mycobacterium tuberculosis must comply with laws, rules, and regulations.
Before start
Prepare buffers

ComponentVolume (ml)
H2O95.8
NaCl (5M)2
Tris-HCl pH 8.3 (1M)1
Triton X-1001
EDTA (0.5M)0.2
Custom Triton Buffer Ph8.3 . To prepare Amount100 mL of the Custom Triton Buffer, simply add each component in the specified amount then add H2O to a final volume of Amount100 mL . Filter sterilize the solution before use. This will result in a final buffer concentration of Concentration100 millimolar (mM) NaCl, Concentration10 millimolar (mM) Tris-HCl; Concentration1 millimolar (mM) EDTA, Concentration1 % (v/v) Triton X-100.

ComponentVolume (ml)
H2O99
Tris-HCl (1M, pH 8)1
EDTA (0.5M)0.02
Low EDTA TE (1X) Ph8 . To prepare Amount100 mL of the Low EDTA Tris Buffer simply add each component in the specified amount then add H2O to a final volume of Amount100 mL . The final buffer has a concentration of
Concentration10 millimolar (mM) Tris-HCl and Concentration01. millimolar (mM) EDTA.

Step case

Sputum sample
18 steps

Prepare Input
Prepare Input
Add four volumes of 100mM dithiothreitol to the sputum sample and vortex for >Duration00:00:30

30s
Incubate at TemperatureRoom temperature for Duration00:15:00

15m
Remove and discard the supernatant, and resuspend the pellet in Amount350 µL of Custom Triton Buffer
Vortex sample and Centrifigation, 00:15:00 , max speed

15m
If it is not possible to do the above in your laboratory.
Use BBL MycoPrepTM (BD) reagent to process the sample according to the manufacturer's instructions with the following modification: Resuspend the sediment in step 8 in the protocol in Amount350 µL of Custom Triton Buffer
If required (i.e., to remove samples for processing outside a BSL-3), decontaminate the sample according to the standard operating procedures of your facility.
Extract DNA
Extract DNA
2m
2m
Transfer the inactivated bacterial suspension to a new well-labeled Starsted screw cap tube containing ~ Amount250 µL of Mini-BeadBeater Zirconia-Silicate Beads, Thikness-0.1 mm

Bead beat the lysate at 6.5m/s for Duration00:00:45 withDuration00:02:00 rest on ice between runs

Repeat for a total of three bead beating cycles

Centrifuge at max speed Centrifigation10000 rcf, Room temperature, 00:02:00 and transfer the supernatant to a new well-labeled tube.
Take care not to transfer beads or cell debris.
2m
Add Amount250 µL (1.2X volume) AMPure XP beads and mix by pipetting up and down 10 times
Incubate at TemperatureRoom temperature for Duration00:05:00
Place on magnetic rack and wait for the solution to clear, ~ Duration00:02:00

Discard the supernatant and wash the beads twice with freshly prepared Concentration70 % (v/v) ethanol without disrupting the beads

Dry the beads briefly, ~ Duration00:02:00 .
Tip: Remove residual EtOH with a p10 pipette

Immediately after the bead pellet becomes opaque, remove the tube from magnetic rack and resuspend in Amount20 µL of Low EDTA Tris Buffer. Ensure all beads are in solution.

Incubate at room temperature for Duration00:05:00

Place on magnetic rack, wait for the solution to become clear ~Duration00:02:00 , and transfer the eluted DNA to a new well-labeled tube