Sep 07, 2022

Public workspaceIntegra Magbead DNA and RNA Extraction for isolated colonies 

DOI
dx.doi.org/10.17504/protocols.io.dm6gpjeqjgzp/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.dm6gpjeqjgzp/v1
Protocol Citation: Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Cristina Tato, Jessica Manning, Vida Ahyong 2022. Integra Magbead DNA and RNA Extraction for isolated colonies . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjeqjgzp/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 02, 2022
Last Modified: September 07, 2022
Protocol Integer ID: 69485
Keywords: Integra, DNA, RNA, Colony, isolated, Extraction
Abstract
This protocol is the process to extract DNA and RNA from isolated colonies. The extracted high-quality DNA or RNA are suitable for Next-Generation Sequencing (NGS).
Guidelines
Adapted from the ZymoBIOMICS MagBead DNA/RNA Kit Manual (Zymo Research, Cat#R2135).
Materials
1. RNase away spray for RNase decontaminants.
ReagentRNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #7002PK

2. ReagentZymoBIOMIC MagBead DNA/RNAZymo ResearchCatalog #R2135
3.Reagent100% Molecular grade ethanol
4.ReagentMolecular Grade Isopropanol
5.ReagentProteinase K w/ Storage buffer 20mg setZymo ResearchCatalog #D3001-2-20
6.ReagentDNase I SetZymo ResearchCatalog #E1010
7.ReagentNuclease-free water AmbionCatalog #AM9932

8. 1ml deep well sterile plate.

9. 2ml deep well sterile plate.

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

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

12. 96S Super Magnet. (ALPAQUA, Cat# A001322)
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


Safety warnings
All steps should be performed at TemperatureRoom temperature .
Perform the 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.
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 of extra air blows out at the end of dispensing steps in plates.




Buffer Preparation
Buffer Preparation
30m
30m
1. Add Amount20 mL isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
2. AddAmount30 mL isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
3. Reconstitute lyophilized Proteinase K at Concentration20 mg/mL with Proteinase K Storage Buffer and mix by vortexing. Use immediately or store at Temperature-20 °C .
4. Reconstitute each vial of lyophilized DNase I with Amount2.25 mL DNase/RNase-Free water in a conical tube.


Note
For each sample to be treated, prepare DNase I Reaction Mix (scale up proportionally):
Add Amount45 µL DNase I (reconstituted) and Amount5 µL DNA Digestion Buffer in a nuclease-free tube.
mix by gentle inversion and place TemperatureOn ice until ready to use.



30m
Make buffer plates prior to starting protocol
Make buffer plates prior to starting protocol
1h
1h
1. Pre-make Lysis Buffer plate with Amount520 µL DNA/RNA Lysis buffer in 1ml deep well plate.
2. Pre-make Beads plate with Amount35 µL ZymoBIOMIC MagBinding Beads into 96 V-well PCR plate.

Note
For the Beads plate, make it immediately before starting, <1h prior to starting the protocol, to ensure the beads are kept in suspension.

3. Pre-make DNA/RNA Wash 1 plate with Amount520 µL MagBead DNA/RNA Wash 1 into 1ml deep well plate. Make it two plates.
4. Pre-make DNA/RNA Wash 2 plate with Amount520 µL MagBead DNA/RNA Wash 2 into 1ml deep well plate. Make it two plates.
5. Pre-make 100% Ethanol plate with Amount1100 µL of 100% Ethanol into a 2ml deep well plate. Make it three plates.
6. Pre-make Prep Buffer plate with Amount520 µL DNA/RNA Prep Buffer into a 1ml deep well plate.
7. Pre-make water plate with Amount60 µL Nuclease-free water in a 96 V-well PCR plate. Make it two plates.
8. Spin all plates down for Duration00:01:00 except for the bead plate. Perform a quick pulse spin down of the bead plate, just enough to get all the liquid down. Centrifuge the rest of the plate at 12 000 rpm for Duration00:01:00 .

1h
Sample preparation and Proteinase K
Sample preparation and Proteinase K
31m
31m
1. Create a plate map so you know which sample you are adding to each well. Add Amount50 µL of isolated colonies samples to plate 1 (leave column 12 for water control).
2. Top up the 1x DNA/RNA Shield to get Amount750 µL .
3. Manually add Amount120 µL of Proteinase K into the 0.2ml 8-strip well.
4. Use multichannel pipet to add Amount10 µL of Proteinase K into each sample and mix (plate 1).
5. Load a set of Integra tips (tip set 1) onto the Integra.
6. Program: Pipet/Mix 250ul, 15 cycles, speed 4. 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 Duration00:30:00 . Keep tips.

31m
Sample binding and washing
Sample binding and washing
35m
35m
7. Program: Pipet 250ul. Add Amount500 µL total of Lysis Buffer to the sample plate (plate 1).
8. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix samples and buffer for Duration00:02:00 . Keep tips.
9. Aliquot Amount35 µL of MagBinding Beads into 96 V-well PCR plate.
10. Program: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended.
11. Program: Pipet 30ul. Add Amount30 µL of MagBinding Beads into the sample plate (plate 1).
12. Program: Pipet/Mix 250ul, 30 cycles, speed 3. Program the Integra to mix the sample and MagBinding Beads plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for Duration00:20:00 .
13. Transfer the plate/tube to the magnetic stand for Duration00:05:00 until beads (DNA) have pelleted, transfer the cleared supernatant (RNA) into a new 96 V-well plate.

35m
DNA Purification (Beads)
DNA Purification (Beads)
45m
45m
14. Change new Integra tips.
15. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL MagBead DNA/RNA Wash 1 into sample plate and mix well.
16. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 1 buffer with the beads. Keep tips.
17. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
18. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
19. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL MagBead DNA/RNA Wash 2 into sample plate and mix well.
20. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 2 buffer with the beads. Keep tips.
21. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
22. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
23. Change new Integra tips.
24. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL 100% Ethanol into sample plate and mix well.
25. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix 100% Ethanol with the beads. Keep tips.
26. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
27. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
28. Repeat step 24.
29. Dry the beads for Duration00:10:00 on the magnetic stand.
30. Change new Integra tips.
31. Program: Pipet 30ul, speed 5. Dispense a total of Amount30 µL nuclease-free water into the sample plate.
32. Program: Pipet/Mix 20ul, 30 cycles, speed 7. Program the Integra to mix nuclease-free water with the beads. Keep tips.
33. Program: Manual Pipet 30ul, speed 3. Transfer the plate to the magnetic stand and pellet the beads for Duration00:05:00 , then aspirate and dispense the eluted DNA to a new 96 V-well plate.
34. Store DNA sample immediately at Temperature-80 °C .

45m
RNA Purification (Supernatant)
RNA Purification (Supernatant)
45m
45m
35. Change the new Integra tip.

36. Program: Pipet 230ul, 3 times, speed 7. Dispense a total of Amount690 µL 100% Ethanol to the supernatant.
37. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix 100% Ethanol with the supernatant. Keep tips.
38. Aliquot Amount35 µL of MagBinding Beads into 96 V-well PCR plate.
39. Program: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended.
40. Program: Pipet 30ul. Add Amount30 µL of MagBinding beads into the sample plate.
41. Program: Pipet/Mix 250ul, 10 cycles, speed 3. Program the Integra to mix the sample and MagBinding beads
plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for Duration00:10:00 .
42. Transfer the plate to the magnetic stand for Duration00:05:00 until beads have pelleted, then discard the cleared supernatant.
43. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL MagBead DNA/RNA Wash 1 into sample plate.
44. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 1 buffer with the beads. Keep tips.
45. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
46. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
47. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL MagBead DNA/RNA Wash 2 into sample plate.
48. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the Wash 2 buffer with the beads. Keep tips.
49. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
50. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
51. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL 100% Ethanol into the sample plate.
52. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix 100% Ethanol with the beads. Keep tips.
53. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
54. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
55. Repeat step 51.
56. DNase I treatment, use multiple channel pipet to transfer Amount50 µL of DNase I Reaction Mix and mix gently for Duration00:10:00 .
57. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of Amount500 µL DNA/RNA Prep Buffer into sample plate.
58. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix the DNA/RNA Prep Buffer with the beads. Keep tips.
59. Place the 96-well magnetic stand underneath the sample plate for Duration00:02:00 until a bead ring forms.
60. Program: Manual Pipet 250ul, 2 times, speed 3. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
61. Repeat step 57 to 60.
62. Program: Pipet 30ul, speed 5. Dispense a total of Amount30 µL nuclease-free water into the sample plate.
63. Program: Pipet/Mix 20ul, 30 cycles, speed 7. Program the Integra to mix nuclease-free water with the beads. Keep tips.
64. Program: Manual Pipet 30ul, speed 3. Transfer the plate to the magnetic stand and pellet the beads for Duration00:05:00 , then aspirate and dispense the eluted RNA to a new 96 V-well plate.
65. Store RNA sample immediately at Temperature-80 °C .

45m