Sep 05, 2022

Public workspaceIntegra Total Nucleic Acid Extraction

DOI
dx.doi.org/10.17504/protocols.io.81wgbydq1vpk/v1
  • 1International Center of Excellence in Research, National Institute of Health, Cambodia;
  • 2Chan Zuckerberg Biohub;
  • 3National Institute of Health
Sophana Chea
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DOI: dx.doi.org/10.17504/protocols.io.81wgbydq1vpk/v1
Protocol Citation: Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Jessica Manning, Vida Ahyong 2022. Integra Total Nucleic Acid Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbydq1vpk/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 01, 2022
Last Modified: September 06, 2022
Protocol Integer ID: 69433
Keywords: Integra, Total Nucleic Acid, DNA, RNA
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).
Guidelines
Adapted from the Quick-DNA/RNA Pathogen MagBead Kit Manual (Zymo Research, Cat# R2145)
Materials
1. RNase away sprays for RNase decontaminants (Thermo Scientific, Cat#7002).
ReagentRNase Away sprayMolecular BioProducts
2. 1ml deep well sterile plate.

3. 2ml deep well sterile plate.

4. Hard-shell PCR Plates 96-well (Bio-Rad, Cat# HSP9601).

5. PCR Plate Seal, foil (Bio-Rad, Cat# MSF1001).

6. Quick-DNA/RNA Pathogen MagBead kit (Zymo Research, Cat# R2145)
ReagentQuick-DNA/RNA Pathogen MagBead kitZymo ResearchCatalog #R2145
7. Molecular-Grade Isopropanol (100% isopropanol).

8. Molecular-Grade Absolute ethanol (100% ethanol).

9. Proteinase K w/ Storage buffer 20mg (Zymo Research, Cat# D3001-2-20).
ReagentProteinase K w/ Storage buffer 20mg setZymo ResearchCatalog #D3001-2-20
10. DNase/RNase-free water
ReagentNuclease-free water AmbionCatalog #AM9932
11.
Equipment
VIAFLO
NAME
96 channel pipette
TYPE
Integra
BRAND
VIAFLO 96
SKU
https://www.integra-biosciences.com/united-kingdom/en/electronic-pipettes/viaflo-96-viaflo-384#tech-info
LINK

12. DNase I (Zymo Research, Cat# E1010)
ReagentDNase I setZymo ResearchCatalog #E1010

13. 96-well magnetic stand
Safety warnings
All steps should be performed at TemperatureRoom temperature .
*NOTE: When reusing tips, make sure to include a bit of extra air aspiration to avoid drops at the bottom of tips when aspirating volumes, and also a bit extra air blows out at the end of dispensing steps in plates.

Perform the TNA extraction in the extraction room separate from the PCR room.
Respect the Laboratory safety guideline for all steps of the protocol.
Wearing PPE is recommended.
Reagent preparation (required with new kit)
Reagent preparation (required with new kit)
15m
15m
1. Add Amount20 mL of isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
2. Add Amount30 mL of isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
3. Add Amount1.2 mL of Proteinase K Storage Buffer per vial to reconstitute the lyophilized Proteinase K at Concentration20 mg/mL
Vortex to dissolve. STORE AT Temperature-20 °C Freezer.

15m
Preparation the buffer plate (before starting protocol)
Preparation the buffer plate (before starting protocol)
1h
1h
1. Pre-make pathogen buffer plate with Amount880 µL Pathogen DNA/RNA buffer in 1ml deep well plate.
2. Pre-make bead plate with Amount25 µL MagBinding beads into 96V-well PCR plate.
*Make immediately before starting, <1h prior to starting the protocol, to ensure the beads are mixed.
3. Pre-make DNA/RNA Wash 1 plate with Amount550 µL Wash 1 buffer into a 1ml deep well plate.
4. Pre-make DNA/RNA Wash 2 plate with Amount550 µL Wash 2 buffer into a 1ml deep well plate.
5. Pre-make 100% ethanol plate with Amount1 mL 100% ethanol into a 2ml deep well plate.
6. Pre-make 80% ethanol plate with Amount600 µL 80% ethanol into a 1ml deep well plate.
7. Pre-make water plate with Amount50 µL DNAase/RNAse-free water in a 96 V-well PCR plate.
8. Pre-make water plate with Amount30 µL DNAase/RNAse-free water in a 96 V-well PCR plate.

Spin all plates down for Duration00:01:00 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 Duration00:01:00 .

1h
Sample preparation and Proteinase K
Sample preparation and Proteinase K
16m
16m
1. Create a plate map so you know which sample you are adding to each well. Add Amount400 µL of your samples to 2ml deep well plate (Plate 1).
2. Manually add Amount65 µL of Proteinase K to each well of 8 well PCR strip tubes. Using a manual multichannel pipet, aliquot Amount4 µL of Proteinase K into each sample (Plate 1).
3. Load a set of Integra tips (tip set 1) onto the Integra.
4. Program: Pipet/Mix 250ul, 15 cycles, speed 10. Program the Integra to pipet Amount250 µL of your samples up and down for Duration00:01:00 (15 cycles), then incubate at TemperatureRoom temperature for Duration00:15:00 .
*Keep tips.

16m
Sample binding and washing
Sample binding and washing
1h 20m
1h 20m
5. Program: Pipet 300ul. add Amount800 µL total of Pathogen DNA/RNA Buffer to the sample plate (Plate 1).
6. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix samples and buffer for Duration00:02:00 . Keep tips.
7. 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.
8. Program: Pipet 20ul. Program the Integra to aspirate Amount20 µL from the MagBinding beads plate. Check that all wells have beads!
9. Program: Pipet/Mix 250ul, 30 cycles x 5 (10 min total), speed 7. Program the Integra to mix the beads with the sample for Duration00:10:00 total. Keep tips.
10. Place the 96-well magnetic stand underneath the sample plate for Duration00:05:00 until a bead ring forms.
11. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Discard tips, load new tips.
12. Remove magnetic stand.
13. Program: Pipet 250ul. Dispense a total of Amount500 µL Wash 1 into the sample plate. Keep tips.
14. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the Wash buffer for Duration00:02:00 total. Keep tips.
15. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until bead ring forms.
16. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
17. Program: Pipet 250ul. Dispense a total of Amount500 µL Wash 2 into the sample plate. Keep tips.
18. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the wash buffer for Duration00:02:00 total. Keep tips.
19. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
20. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
21. Remove magnetic stand.
22. Program: Pipet 250ul. Dispense a total of Amount500 µL 100% Ethanol into the sample plate.
23. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with Ethanol for Duration00:02:00 t total. Keep tips.
24. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
25. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate.
26. Remove magnetic stand.
27. Repeat steps 22 to 26 for another round of Ethanol wash. Discard tips after 2nd Ethanol wash. Load new tips.
28. Keep the sample plate on the magnetic stand after the final Ethanol wash until the beads are dry (~Duration00:10:00 ).
29. Program: Pipet/Mix 33ul, 30 cycles, speed 7. Pipet Amount33 µL of Nuclease-free water (Heat the water at Temperature55 °C for Duration00:10:00 before elution) into the dried beads and mix the beads for Duration00:02:00 total. Keep tips.
30. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
31. Program: Manual Pipet 30ul. Pipet Amount30 µL from the sample plate and dispense into a new 96 V-bottom PCR plate.
32. Store TNA sample immediately at Temperature-80 °C .

1h 20m
DNase treatment post-TNA purification
DNase treatment post-TNA purification
15m
15m
1. Aliquot SPRI beads (well mixed and resuspended) Amount50 µL into a new 96 V-bottom PCR plate.
2. Prepare DNase I solution
a. Add Amount275 µL DNase/RNase-free water to reconstitute lyophilized DNase I and mix.
3. Aliquot Amount20 µL of TNA into a new 96 V-bottom PCR plate.
4. Prepare DNase master mix:
a. 1x rxn = 20ul sample + 2.5ul DNase I + 2.5ul Digestion Buffer.
b. Using a manual multichannel pipet, aliquot Amount5 µL of DNase I master mix to each sample and mix.
c. Program: Pipet/Mix 20ul, 20 cycles, speed 7.
5. Incubate Duration00:15:00 TemperatureRoom temperature

15m
SPRI bead clean-up
SPRI bead clean-up
30m
30m
1. Program: Pipet/Mix 45ul, 20 cycles, speed 7. Add Amount45 µL SPRI beads (1.8x ratio) to the DNase I treated sample and mix by pipetting.
2. Incubate Duration00:05:00 at TemperatureRoom temperature .
3. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
4. Program: Manual Pipet 100ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
5. Program: Pipet 200ul. (1st Ethanol Wash). Aspirate and dispense Amount200 µL of freshly made 80% Ethanol into sample plate.
6. Incubate for Duration00:01:00 at TemperatureRoom temperature .
7. Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
8. Program: Pipet 200ul. (2nd Ethanol Wash). Aspirate and dispense Amount200 µL of freshly made 80% Ethanol into sample plate.
9. Incubate for Duration00:01:00 at TemperatureRoom temperature .
10. Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
11. Incubate for Duration00:05:00 at TemperatureRoom temperature until beads are dry.
12. Remove plate from magnetic stand.
13. Program: Pipet/Mix 22ul, 10 cycles, speed 7. AddAmount22 µL DNase/RNase-free water to each well and mix beads.
14. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
15. Program: Manual Pipet 20ul. Aspirate Amount20 µL of purified TNA sample to a new 96 V-bottom PCR plate.
16. Store sample at Temperature-80 °C immediately.

30m