TITLE: Integra Magbead DNA and RNA Extraction for isolated colonies 
AUTHORS: Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Cristina Tato, Jessica Manning, Vida Ahyong

[DESCRIPTION]
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).

[STEPS]
SECTION: Buffer Preparation
1. 1. Add 20 mL isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
2. Add30 mL isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
3. Reconstitute lyophilized Proteinase K at 20 mg/mL with Proteinase K Storage Buffer and mix by vortexing. Use immediately or store at -20 °C .
4. Reconstitute each vial of lyophilized DNase I with 2.25 mL DNase/RNase-Free water in a conical tube. 


 


SECTION: Make buffer plates prior to starting protocol
2. 1. Pre-make Lysis Buffer plate with 520 µL DNA/RNA Lysis buffer in 1ml deep well plate.
2. Pre-make Beads plate with 35 µL ZymoBIOMIC MagBinding Beads into 96 V-well PCR plate.

 

3. Pre-make DNA/RNA Wash 1 plate with 520 µL MagBead DNA/RNA Wash 1 into 1ml deep well plate. Make it two plates.
4. Pre-make DNA/RNA Wash 2 plate with 520 µL MagBead DNA/RNA Wash 2 into 1ml deep well plate. Make it two plates.
5. Pre-make 100% Ethanol plate with 1100 µL of 100% Ethanol into a 2ml deep well plate. Make it three plates.
6. Pre-make Prep Buffer plate with 520 µL DNA/RNA Prep Buffer into a 1ml deep well plate.
7. Pre-make water plate with 60 µL Nuclease-free water in a 96 V-well PCR plate. Make it two plates.
8. Spin all plates down for 1 min 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 1 min .

SECTION: Sample preparation and Proteinase K
3. 1. Create a plate map so you know which sample you are adding to each well. Add 50 µL of isolated colonies samples to plate 1 (leave column 12 for water control).
2. Top up the 1x DNA/RNA Shield to get 750 µL .
3. Manually add 120 µL of Proteinase K into the 0.2ml 8-strip well.
4. Use multichannel pipet to add 10 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 250 µL of your samples up and down for 1 min (15 cycles), then incubate at Room temperaturefor 30 min . Keep tips.

SECTION: Sample binding and washing
4. 7. Program: Pipet 250ul. Add 500 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 2 min .       Keep tips.
9. Aliquot 35 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 30 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 20 min .
13. Transfer the plate/tube to the magnetic stand for 5 min until beads (DNA) have pelleted, transfer the cleared supernatant (RNA) into a new 96 V-well plate.

SECTION: DNA Purification (Beads)
5. 14. Change new Integra tips.
15. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500 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 2 min 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 500 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 2 min 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 500 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 2 min 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 10 min  on the magnetic stand.
30. Change new Integra tips.
31. Program: Pipet 30ul, speed 5. Dispense a total of 30 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 5 min , then aspirate and dispense the eluted DNA to a new 96 V-well plate.
34. Store DNA sample immediately at -80 °C .

SECTION: RNA Purification (Supernatant)
6. 35. Change the new Integra tip.

36. Program: Pipet 230ul, 3 times, speed 7. Dispense a total of 690 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 35 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 30 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 10 min .
42. Transfer the plate to the magnetic stand for 5 min until beads have pelleted, then discard the cleared supernatant.
43. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500 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 2 min 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 500 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 2 min 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 500 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 2 min 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 50 µL  of DNase I Reaction Mix and mix gently for 10 min .
57. Program: Pipet 250ul, 2 times, speed 7. Dispense a total of 500 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 2 min 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 30 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 5 min , then aspirate and dispense the eluted RNA to a new 96 V-well plate.
65. Store RNA sample immediately at -80 °C .