Integra Total Nucleic Acid Extraction

Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Jessica Manning, Vida Ahyong

Published: 2022-09-05 DOI: 10.17504/protocols.io.81wgbydq1vpk/v1

Abstract

This SOP is the process of extracting Total Nucleic Acid (TNA) from Sera and/or Nasopharyngeal or Nasal swabs. The isolated high-quality nucleic acids are suitable for Next-Generation Sequencing (NGS).

Steps

Reagent preparation (required with new kit)

Add 20mL of isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
Add 30mL of isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
Add 1.2mL of Proteinase K Storage Buffer per vial to reconstitute the lyophilized Proteinase K at 20mg/mL

Vortex to dissolve. STORE AT -20°C Freezer.

Preparation the buffer plate (before starting protocol)

Pre-make pathogen buffer plate with 880µL Pathogen DNA/RNA buffer in 1ml deep well plate.
Pre-make bead plate with 25µL MagBinding beads into 96V-well PCR plate.

*Make immediately before starting, <1h prior to starting the protocol, to ensure the beads are mixed.

Pre-make DNA/RNA Wash 1 plate with 550µL Wash 1 buffer into a 1ml deep well plate.
Pre-make DNA/RNA Wash 2 plate with 550µL Wash 2 buffer into a 1ml deep well plate.
Pre-make 100% ethanol plate with 1mL 100% ethanol into a 2ml deep well plate.
Pre-make 80% ethanol plate with 600µL 80% ethanol into a 1ml deep well plate.
Pre-make water plate with 50µL DNAase/RNAse-free water in a 96 V-well PCR plate.
Pre-make water plate with 30µL DNAase/RNAse-free water in a 96 V-well PCR plate.

Spin all plates down for 0h 1m 0s except the bead plate. Perform a quick pulse spin down of the bead plate, just enough to get all liquid down. Centrifuge the rest of the plates at 12 000 rpm for 0h 1m 0s .

Sample preparation and Proteinase K

Create a plate map so you know which sample you are adding to each well. Add 400µL of your samples to 2ml deep well plate (Plate 1).
Manually add 65µL of Proteinase K to each well of 8 well PCR strip tubes. Using a manual multichannel pipet, aliquot 4µL of Proteinase K into each sample (Plate 1).
Load a set of Integra tips (tip set 1) onto the Integra.
Program: Pipet/Mix 250ul, 15 cycles, speed 10. Program the Integra to pipet 250µL of your samples up and down for 0h 1m 0s (15 cycles), then incubate at 25Room temperature for 0h 15m 0s .

*Keep tips.

Sample binding and washing

Program: Pipet 300ul. add 800µL total of Pathogen DNA/RNA Buffer to the sample plate (Plate 1).
Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix samples and buffer for 0h 2m 0s . Keep tips.
Program: Pipet/Mix 20ul, 20 cycles. Program the Integra to mix the MagBinding beads plate so the beads are fully resuspended. If beads are not fully to the bottom of theplate, perform a short pulse spin.
Program: Pipet 20ul. Program the Integra to aspirate 20µL from the MagBinding beads plate. Check that all wells have beads!
Program: Pipet/Mix 250ul, 30 cycles x 5 (10 min total), speed 7. Program the Integra to mix the beads with the sample for 0h 10m 0s total. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 5m 0s until a bead ring forms.
Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.

Discard tips, load new tips.

Remove magnetic stand.
Program: Pipet 250ul. Dispense a total of 500µL Wash 1 into the sample plate. Keep tips.
Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the Wash buffer for 0h 2m 0s total. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until bead ring forms.
Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
Program: Pipet 250ul. Dispense a total of 500µL Wash 2 into the sample plate. Keep tips.
Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the wash buffer for 0h 2m 0s total. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
Remove magnetic stand.
Program: Pipet 250ul. Dispense a total of 500µL 100% Ethanol into the sample plate.
Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with Ethanol for 0h 2m 0s total. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate.
Remove magnetic stand.
Repeat steps 22 to 26 for another round of Ethanol wash. Discard tips after 2nd Ethanol wash. Load new tips.
Keep the sample plate on the magnetic stand after the final Ethanol wash until the beads are dry (~0h 10m 0s ).
Program: Pipet/Mix 33ul, 30 cycles, speed 7. Pipet 33µL of Nuclease-free water (Heat the water at 55°C for 0h 10m 0s before elution) into the dried beads and mix the beads for 0h 2m 0s total. Keep tips.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 30ul. Pipet 30µL from the sample plate and dispense into a new 96 V-bottom PCR plate.
Store TNA sample immediately at -80°C .

DNase treatment post-TNA purification

Aliquot SPRI beads (well mixed and resuspended) 50µL into a new 96 V-bottom PCR plate.
Prepare DNase I solution

a. Add 275µL DNase/RNase-free water to reconstitute lyophilized DNase I and mix.
Aliquot 20µL of TNA into a new 96 V-bottom PCR plate.
Prepare DNase master mix:

a. 1x rxn = 20ul sample + 2.5ul DNase I + 2.5ul Digestion Buffer.

b. Using a manual multichannel pipet, aliquot 5µL of DNase I master mix to each sample and mix.

c. Program: Pipet/Mix 20ul, 20 cycles, speed 7.
Incubate 0h 15m 0s Room temperature

SPRI bead clean-up

Program: Pipet/Mix 45ul, 20 cycles, speed 7. Add 45µL SPRI beads (1.8x ratio) to the DNase I treated sample and mix by pipetting.
Incubate 0h 5m 0s at Room temperature .
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 100ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
Program: Pipet 200ul. (1st Ethanol Wash). Aspirate and dispense 200µL of freshly made 80% Ethanol into sample plate.
Incubate for 0h 1m 0s at Room temperature .
Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
Program: Pipet 200ul. (2nd Ethanol Wash). Aspirate and dispense 200µL of freshly made 80% Ethanol into sample plate.
Incubate for 0h 1m 0s at Room temperature .
Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
Incubate for 0h 5m 0s at Room temperature until beads are dry.
Remove plate from magnetic stand.
Program: Pipet/Mix 22ul, 10 cycles, speed 7. Add22µL DNase/RNase-free water to each well and mix beads.
Place the 96-well magnetic stand underneath the sample plate for 0h 2m 0s until a bead ring forms.
Program: Manual Pipet 20ul. Aspirate 20µL of purified TNA sample to a new 96 V-bottom PCR plate.
Store sample at -80°C immediately.