TITLE: Automation Protocol for High-Efficiency and High-Quality Genomic DNA Extraction from Saccharomyces Cerevisiae
AUTHORS: Nina Alperovich, Benjamin M. Scott, David Ross

[DESCRIPTION]
Here, we describe a protocol for automated extraction of genomic DNA (gDNA) from yeast liquid culture and colonies. The protocol uses a Hamilton NGS-STAR automated liquid handler equipped with thermal-regulated microplate shakers. We have tested this automated protocol with 1.5 mL aliquots of liquid culture (OD600= 1) to demonstrate that it can provide high-quality gDNA at high efficiency. The protocol can also be implemented with manual pipetting, and/or to extract gDNA from larger volumes of culture by proportionally increasing the volumes of reagents and using an appropriate magnet separation rack. 

[STEPS]
SECTION: Preparation of Zymolyase Stock
1. Prepare stock of Zymolyase 20T by dissolving to a concentration of 1 U/µL in 1x PBS.
SECTION: Preparation of Zymolyase Stock
2. Divide Zymolyase solution into 800 µL aliquots and store at -20 °C until use.
SECTION: Preparation of 2-Mercaptoethanol Stock
3. Prepare 0.001 % stock of 2-Mercaptoethanol by diluting 1000-fold into 1x PBS.
SECTION: Preparation of 2-Mercaptoethanol Stock
4. Store at 4 °C until use.
SECTION: Preparation of Digestion Master Mix
5. Add 22.72 mL 1x PBS
SECTION: Preparation of Digestion Master Mix
6. Add one aliquot (800 µL) of Zymolyase Stock, 1 U/µL
SECTION: Preparation of Digestion Master Mix
7. Add 160 µL 0.001 % 2-Mercaptoethanol
SECTION: Preparation of Digestion Master Mix
8. Add 320 µL RNase A/T1 mix
SECTION: Preparation of Digestion Master Mix
9. Prepare fresh before each use
SECTION: Preparation of 4x Lysate Buffer
10. Add 30 mL Nuclease-Free Water.
SECTION: Preparation of 4x Lysate Buffer
11. Add 60 mL 20% SDS
SECTION: Preparation of 4x Lysate Buffer
12. Add 4 mL 0.5 mol/L EDTA.
SECTION: Preparation of 4x Lysate Buffer
13. Add 6 mL 1 mol/L TRIS-HCL, pH 8.0
SECTION: Preparation of 4x Lysate Buffer
14. Store at 4 °C until use.
SECTION: Preparation of Magnetic Beads Stock
15. Vortex Speed-Bead Magnetic beads to resuspend
SECTION: Preparation of Magnetic Beads Stock
16. Divide Speed-Bead Magnetic beads into 1 mL aliquots, each in in a 1.7 mL Microcentrifuge Tube.
SECTION: Preparation of Magnetic Beads Stock
17. Place micro-centrifuge tubes containing magnetic beads solutions on a 12-Tube Magnetic Separation Rack until beads are drawn to the magnet and solutions are clear.
SECTION: Preparation of Magnetic Beads Stock
18. Remove supernatant.
SECTION: Preparation of Magnetic Beads Stock
19. Add 1 mL 1x TE Buffer to each micro-centrifuge tube containing beads, remove from magnetic stand, and mix by vortex.
SECTION: Preparation of Magnetic Beads Stock
20. Repeated steps 17-19 two additional times.
SECTION: Preparation of Magnetic Beads Stock
21. Store bead stock in the dark at 4 °C, then mix bead stock well by vortexing before use.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
22. Add 9 g PEG-8000 to a new 50 mL Conical Tube.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
23. Add 10 mL 5 mol/L Sodium Chloride.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
24. Add 500 µL 1 mol/L Tris-HCl, pH8.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
25. Add 500 µL 0.5 mol/L EDTA.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
26. Fill Conical Tube to the 40 mL mark with Nuclease-Free Water, then vortex to mix every 1-2 minutes until PEG goes into solution.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
27. Add 27.5 µL Tween-20 and mix gently by inverting 4-6 times.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
28. Vortex Magnetic Beads Stock (from previous section) to mix thoroughly.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
29. Add Magnetic Bead Stock:
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
30. Fill Conical Tube to the 50 mL mark with Nuclease-Free Water, and gently mix until solution is a uniform brown color.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
31. Wrap in foil (or place in dark container) and store at 4 °C, mix well by vortexing before use.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
29.1. For extraction of gDNA from liquid culture, add 3 mL Magnetic Beads Stock to the 50 mL Conical Tube containing the PEG/NaCl solution.
SECTION: Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
29.2. For extraction of gDNA from yeast colonies, add 1 mL Magnetic Beads Stock to the 50 mL Conical Tube containing the PEG/NaCl solution.
SECTION: Preparation of yeast liquid culture for extraction
32. Aliquot 1.5 mL (OD600=1) in 1-3 columns of 96-well Spin Plate using automated multichannel pipettes.
SECTION: Preparation of yeast liquid culture for extraction
33. Centrifuge at 3700 g for 10 min at 10 °C.
SECTION: Preparation of yeast liquid culture for extraction
34. Remove supernatant from each well using automated liquid handler.
SECTION: Preparation of yeast liquid culture for extraction
35. Seal Spin Plate using Aluminum Foil Lid and Rubber Roller.
SECTION: Preparation of yeast liquid culture for extraction
36. Store at -20 °C.
SECTION: Preparation of yeast liquid culture for extraction
37. Defrost at 4 °C for 30-60 minutes before continuing with extraction.
SECTION: Preparation of yeast liquid culture for extraction
34.1. Use modified labware definition or other constraints on automated pipetting to set a minimum distance of 2.5 mm between the bottom of each well and the tip of each pipette. 
SECTION: Preparation of yeast liquid culture for extraction
34.2. The resulting volume of the remaining cell pellet and residual liquid should be approximately 50 µL.
SECTION: Preparation of yeast colonies for extraction
38. Pick up colonies from agar plate and resuspend each in 50 μL of PBS in 1-3 columns of 96-well Spin Plate.
SECTION: Preparation of yeast colonies for extraction
39. Proceed with DNA extraction from resuspended colonies as described below.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
40. Pipette 315 µL of Digestion Master Mix into each well containing a cell pellet or resuspended colony; mix 30 times by repeated aspiration and dispense.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
41. Transfer 315 μL from each well containing resuspended cell sample to a well in a new Reaction Plate located on a thermal-regulated microplate shaker.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
42. Incubate at 37 °C for 25 minutes while shaking at 400 rpm.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
43. Pipette 100 µL of 4x Lysate Buffer to each well and mix 3 times by repeated aspiration and dispense.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
44. Incubate at 75°C for 10 minutes.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
45. Move the Reaction Plate to a microplate shaker at room temperature.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
46. Pipette 200 µL Magnetic Bead/PEG-NaCl Binding Buffer into each well.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
47. Incubate at room temperature for 7 minutes while shaking at 300 rpm.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
48. Move the Reaction Plate to a 96-well magnet base to pull the magnetic beads out of suspension
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
49. Wait for 5 minutes.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
50. Remove and discard supernatant from each sample (approximately 615 μL).
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
51. Add 600 µL 80 % Ethanol to each well.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
52. Wait for 1 minute.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
53. Remove and discard the ethanol supernatant.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
54. Add 300 µL 80 % Ethanol to each well.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
55. Wait for 1 minute.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
56. Remove and discard the ethanol supernatant.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
57. Allow magnetic bead pellets to dry for 5 minutes at room temperature.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
58. Move the Reaction Plate from magnet base to an automated thermal-regulated microplate shaker with temperature set to 60 °C.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
59. Add 100 µL Nuclease-Free Water to each well (note: can also use EB)
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
60. Resuspend beads by repeated aspiration and dispense (12 times).
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
61. Incubate at 60 °C for 10 s while shaking at 1000 rpm.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
62. Incubate at 60 °C for 7 minutes while shaking at 400 rpm.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
63. Move the Reaction Plate back to the magnet base.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
64. Wait for 5 minutes.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
65. Transfer the supernatant from each well to a new, clean well in the same plate (still on the magnet base).
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
66. Wait for 5 minutes.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
67. Transfer the supernatant with extracted DNA from each well to a well in the Elution plate.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
68. Cover the Elution plate containing the extracted DNA samples with a Universal Lid.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
46.1. Mix Magnetic Bead/PEG-NaCl Binding Buffer well by repeated aspiration and dispense before each transfer.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
46.2. After dispensing beads into each well, mix beads with cell lysate with one aspiration and dispense.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
46.3. During this step, the ratio of Magnetic Bead/PEG-NaCl Binding Buffer to the lysed sample volume is 0.48 (200 µL : 415 µL). Consequently, because of the relatively low PEG concentration in the mixture, only high molecular weight chromosomal DNA will bind to the beads.
SECTION: Yeast genomic DNA extraction. The following steps are implemented with an automated liquid handler, but can also be implemented with manual pipetting
56.1. After removing most of the supernatant with large-volume tips, use smaller-volume tips (e.g., 50 µL) to remove any residual supernatant from the bottom of each well.