Feb 27, 2023

Public workspaceCOAST Biostic gDNA extraction using vacuum manifold

Forked from a private protocol
This protocol is a draft, published without a DOI.
  • 1UCSF
holly.steininger
Icon indicating open access to content
QR code linking to this content
Protocol Citation: holly.steininger 2023. COAST Biostic gDNA extraction using vacuum manifold. protocols.io https://protocols.io/view/coast-biostic-gdna-extraction-using-vacuum-manifol-cp5rvq56
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: February 27, 2023
Last Modified: February 27, 2023
Protocol Integer ID: 77713
Abstract
COAST Extraction Protcol - HMS
Materials
  1. Qiagen QIAamp Biostic Bacteremia DNA kit (for bead tubes and reagents only), cat# 12240-50
  2. Qiagen DNeasy UltraClean 96 Microbial Kit (for DNA columns only), cat# 10196-4
  3. USA Scientific 2.0 ml deep 96-well PP plate, sterile, cat #1896-2110
  4. Vacuum manifold for 96 well plate
Sample prep
Sample prep
2h 16m 10s
2h 16m 10s
Place tubes on bin of ice for approximately Duration01:00:00 to thaw sample fully. Record all information present as you move the tubes.

1h
Aliquot Amount100 µL of IRS solution into each well a 2ml deep 96 well plate and seal with foil
Aliquot Amount1 mL BB solution into each well of a 2ml deep 96 well plate and seal with foil
Aliquot Amount700 µL EB solution in 8 2mL tubes
Label 96 Powerbead tubes 1-93 (or sample number) and 3 control tubes on lid and side of the tube



DNA isolation
DNA isolation
51m 10s
51m 10s
Heat MBL solution at Temperature55 °C for Duration00:05:00 to Duration00:10:00 . Using a multichannel and a reservoir transfer Amount450 µL MBL solution to Amount2 mL PowerBead Tube. Add as much sample as possible per tube at a maximum amount of 1.3 mL (650x2).
Create 3 NTCs per extraction batch
15m
Vortex forDuration00:00:10 to mix and place in a Temperature70 °C heat block or water bath for Duration00:15:00
due to limited heat blocks you have to heat in batches of 48
15m 10s
Secure the PowerBead Tube horizontally using the Vortex Adapter tube holder for the vortex (cat. no. 13000-V1-24). Vortex at maximum speed for Duration00:10:00 .
due to limited vortex attachments you have to vortex in batches of 48
10m
Centrifuge the PowerBead Tube to pellet debris at Centrifigation10.000 x g, 00:01:00 .
1m
Add supernatant to the 2 ml deep well 96 well plate with pre aliquoted Amount100 µL of Solution IRS, seal, and vortex to mix. Incubate for Duration00:05:00 at room temperature. Note: Longer incubation in Solution IRS does not affect DNA yield or purity (sample may be incubated up to 10 min in Solution IRS).

5m
Centrifuge at Centrifigation3.750 rpm, 00:03:00 .
Note
12/19/22 MB I did this centrifuge step at 3,700 g


3m
Transfer the supernatant with a multichannel pipette to 2ml deep 96 well plate containing BB solution. Pipette or pulse vortex to mix. Briefly centrifuge to collect any liquid from the top of the lid.

Place 96 well DNA column plate on a vacuum manifold and load Amount800 µL of lysate to the wells. Turn on vacuum and allow all liquid to pass through. Repeat this step until all the lysate has been loaded onto the DNA column (usually only x2).

Wash by adding Amount500 µL of Solution CB. Turn on vacuum and allow all liquid to pass through.

Wash with another Amount500 µL of Solution CB and Turn on vacuum and allow all liquid to pass through

Continue to allow vacuum to dry the DNA Column membrane. Approximately Duration00:02:00

2m
Remove well plate and place over a 96-well semi skirted plate.
Elute by adding Amount50 µL of Solution EB directly in the center of the membrane. Allow sit at room temperature for up to 5 min to maximize the elution.

Centrifuge the stacked plates in the centrifuge at Centrifigation3.750 x g, 00:02:00 to elute DNA.
This step the membrane+ plate looks very suspect I covered the membrane in foil and used foil to

secure the membrane to the semi skirt plate.

Note
12/19/22 MB I did this centrifuge step at 3,700 g

2m
Discard DNA column plate and aliquot Amount2 µL of eluted DNA into a second semi skirted 96 well plate.

Seal both plates containing Amount48 µL of eluted DNA and the other containing Amount2 µL with foil and store at Temperature-20 °C
Note: We recommend storing DNA frozen (–20° to –80°C) as Solution EB does not contain EDTA.