TITLE: DNA Extraction 
AUTHORS: izabel.stohel@pnnl.gov

[DESCRIPTION]
A list of all protocols used, from Zymo Research with slight modification. Additionally, protocols for measurement of purity and concentration are listed as well. 

D6010 is used when there are only a handful of samples and we use tubes, with the corresponding clean and concentrate protocol being D4013 . D4302, D4306, D4308 is the protocol used when there are a large number of samples and we are using a plate, and the corresponding clean and concentrate protocol is D4066. For bottoms of cores that have less DNA, protocol D6110 has been effective. 

[STEPS]
24. The filter may be discarded and the eluted DNA is now in the 1.5mL microcentrifuge tube
SECTION: Measuring Concentration and Purity with Nanodrop
26. Select dsDNA and follow the prompts from the Nanodrop.
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
1. Print Labels for 1.5mL tubes (“Monet, Sample ID, Core Location, DNA")
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
2. Measure 200mg of soil in ZR Bashing Bead Lysis Tube, do not exceed 200mg as to not overwhelm the tube
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
3. Remove all the lids and add 750uL of ZymoBIOMICS lysis solution
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
4. Recap and put in a bead beater for 10 minutes
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
5. Centrifuge ZR Bashing Bead Lysis Tube in a microcentrifuge at 16,000 x g for 1 minute
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
6. Pipette 350uL of the solution in Zymo-Spin III-F filter, placed in a collection tube. Pipette the remaining solution into another Zymo-Spin III-F filter with collection tube. Now you have two sets of DNA from one original sample that can be combined later
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
7. Centrifuge at 8,000 x g for one minute (If you get a pellet, pipette supernatant to a fresh tube)
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
8. Discard the filter
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
9. Add 800uL of Genomic Lysis Buffer and 400uL of ethanol to the filtrate. Mix well
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
10. Transfer the solution to a Zymo-Spin IICR filter and collection tube. (Note: You cannot fit the entire solution in the filter, you will need to do the following steps at least twice:)
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
11. Centrifuge at 10,000 x g for 1 minute. Discard flow through
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
12. Add the rest of the solution from step 9 and repeat step 11
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
13. Put the filter in a new collection tube
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
14. Add 200uL of DNA pre-wash buffer to the Zymo-Spin IICR Column and centrifuge at 10,000 x g for 1 minute
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
15. Add 500uL of g-DNA wash buffer to the Zymo-Spin IICR Column
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
16. Centrifuge at 10,000 x g for 1 minute. Discard flow through
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
17. Repeat step 16
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
18. Get a new collection tube for the filter and change gloves
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
19. Add 100uL of elution buffer to the filter and let incubate at room temperature for 1 minute
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
20. Centrifuge at 10,000 x g for 1 minute
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
21. Add 600uL of prep solution to a new III-HRC filter with a collection tube and let incubate for 3 minutes. Then centrifuge at 8000 x g for 3 minutes. Discard flow through.
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
22. Put the III-HRC filter into a 1.5mL microcentrifuge tube with cap.
SECTION: DNA Extraction Protocol by Hand: Quick DNA Fecal/Soil Microbe Miniprep Kit D6010
23. Discard the filter from step 19 and pipette the eluted DNA to the prepared III-HRC filter
25. Centrifuge at 16,000 x g for 3 minutes
SECTION: Measuring Concentration and Purity with Nanodrop
26.1. Use 2uL of nuclease free water as the blank before you begin measuring samples
SECTION: Measuring Concentration and Purity with Nanodrop
26.2. Once the blank has been calibrated use 2uL of sample to measure the concentration and purity
SECTION: Measuring Concentration and Purity with Qubit
27. Set up two standards, (Note: keep buffer away from the light as much as possible)
27.1. Measure 190uL of buffer and 10uL of standard in Qubit measurement tube
28. Vortex the tubes briefly
29. Incubate in the dark for two minutes
30. Measure 198uL of buffer and 2uL of sample, vortex briefly and incubate in the dark for two minutes
31. Follow Qubit prompts and measure standards and then sample
SECTION: DNA Clean and Concentrater-5 Protocol D4013
32. Add 200uL of DNA Binding Buffer to the eluted DNA
SECTION: DNA Clean and Concentrater-5 Protocol D4013
33. Transfer the mixture to a Zymo-Spin column in a collection tube. If you have multiple sets of DNA, combine them in this step
SECTION: DNA Clean and Concentrater-5 Protocol D4013
34. Centrifuge for 30 seconds at 16000 x g and discard flow through
SECTION: DNA Clean and Concentrater-5 Protocol D4013
35. Add 200uL of DNA Wash Buffer centrifuge for 30 seconds at 16000 x g then repeat this step again.
SECTION: DNA Clean and Concentrater-5 Protocol D4013
36. Put the filter in a 1.5mL microcentrifuge tube with cap
SECTION: DNA Clean and Concentrater-5 Protocol D4013
37. Add 100uL of DNA Elution Buffer to the filter, let incubate for 1 minute at room temperature
SECTION: DNA Clean and Concentrater-5 Protocol D4013
38. Centrifuge at 16000 x g for 1 minute. Eluted DNA is now ready for use
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
39. Print Labels for 1.5mL tubes (“Monet, Sample ID, Core Location, DNA) place on ZR BashingBead Lysis Rack
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
40. Measure 200mg of soil in ZR Bashing Bead Lysis Tube, do not exceed 200mg as to not overwhelm the tube
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
41. Remove all the lids and add 750uL of ZymoBIOMICS lysis solution
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
42. Recap and put in a bead beater for 10 minutes
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
43. Centrifuge ZR Bashing Bead Lysis Tube in a microcentrifuge at 16000 x g for 1 minute
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
44. Add 600uL of ZymoBIOMICS MagBinding Buffer and 25uL of the beads to 3 96 well plates. Mix well
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
45. Add 200uL of supernatant to each 96 well plate so you have 3 sets of the sample
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46. Load plate in the Eppendorf epMotion and begin protocol: (Note: since there are three plates, epMotion can only one run one a day, so this will take three days to complete)
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
47. Centrifuge plates at 3486 x g for 3 minutes before you start
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
48. Add 200uL of ChIP DNA Binding Buffer to each volume of DNA sample
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
49. Mix well and transfer mixtures to the provided Zymo-Spin I-96-XL Plate mounted on a collection plate and centrifuge for 5 minutes at 3486 x g
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
50. Repeat step 49 with duplicate samples
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
51. Add 200uL of DNA Wash Buffer and centrifuge for 5 minutes at 3486 x g
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
52. Centrifuge again to ensure all solution has filtered through to the collection plate
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
53. Put Zymo-Spin I-96-XL Plate on an Elution Plate and add 50uL of Elution Buffer
SECTION: ZR-96 Genomic DNA Clean and Concentrator-5 Kit Protocol D4066
54. Centrifuge for 5 minutes at 3486 x g to elute the DNA.
SECTION: 50mL Tube DNA Extraction Protocol D6110
55. Print Labels for 50mL tubes (“Monet, Sample ID, Core Location, DNA). Add 2.5-5g of soil to the bead/filter chamber of a ZR BashingBead Lysis/Filtration Tube
SECTION: 50mL Tube DNA Extraction Protocol D6110
56. Add 6mL of BashingBead buffer
SECTION: 50mL Tube DNA Extraction Protocol D6110
57. Shake on bead beater for ten minutes
SECTION: 50mL Tube DNA Extraction Protocol D6110
58. Centrifuge at 3486 x g for five minutes
SECTION: 50mL Tube DNA Extraction Protocol D6110
59. Transfer the supernatant to a clean 50mL tube, there is likely a pellet
SECTION: 50mL Tube DNA Extraction Protocol D6110
60. Add 18mL of Genomic Lysis Buffer to supernatant and mix well
SECTION: 50mL Tube DNA Extraction Protocol D6110
61. Filter entire mixture using Zymo-Spin V-E Column / Zymo-Midi Filter with a new 50mL tube, Centrifuge at 2000 x g for five minutes
SECTION: 50mL Tube DNA Extraction Protocol D6110
62. Repeat step 61, because the mixture won’t all fit the first time
SECTION: 50mL Tube DNA Extraction Protocol D6110
63. Centrifuge at 2000 x g for five minutes once more to ensure the solution has entirely filtered
SECTION: 50mL Tube DNA Extraction Protocol D6110
64. Transfer the Zymo-Spin V-E Column to a collection tube and spin at 10000 x g for 1 minute in a microcentrifuge. (The filter disconnects from the column)
SECTION: 50mL Tube DNA Extraction Protocol D6110
65. Add 300uL of DNA Pre-Wash Buffer to the filter and spin at 10000 x g for 1 minute and discard flow through
SECTION: 50mL Tube DNA Extraction Protocol D6110
66. Add 400uL of g-DNA wash Buffer to the column and centrifuge at 10000 x g for 1 minute, discard flow through
SECTION: 50mL Tube DNA Extraction Protocol D6110
67. Repeat step 66
SECTION: 50mL Tube DNA Extraction Protocol D6110
68. Place Zymo-Spin III-HRC in a clean collection tube and add 600uL of prep solution. Let incubate for 3 minutes then centrifuge at 8000 x g for 3 minutes
SECTION: 50mL Tube DNA Extraction Protocol D6110
69. Transfer the Zymo-Spin III-HRC filter to a 1.5mL microcentrifuge tube
SECTION: 50mL Tube DNA Extraction Protocol D6110
70. Transfer the Zymo-Spin V-E Column to a clean collection tube and add 150uL of Elution Buffer directly to the column matrix, let incubate for a minute and then centrifuge at 10000 x g for 1 minute
SECTION: 50mL Tube DNA Extraction Protocol D6110
71. Transfer the eluted DNA to the prepared III-HRC filter and centrifuge at 16000 x g for 3 minutes. Eluted DNA is ready for use. 
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.1. a.       Transfer the 96-well block to a magnetic stand until beads pellet, then aspirate and discard the supernatant. Remove the 96-Well Block from the magnetic stand
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.2. b.       Dispense 500 µl of ZymoBIOMICS™ MagBinding Buffer and mix well by pipette
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.3. c.       Transfer the 96-well block to a magnetic stand until beads pellet, then aspirate and discard the supernatant. Remove the 96-Well Block from the magnetic stand.
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.4. d.       Dispense 500 µl of ZymoBIOMICS™ MagWash 1 and mix well by pipette
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.5. e.       Transfer the 96-well block to a magnetic stand until beads pellet, then aspirate and discard the supernatant. Remove the 96-Well Block from the magnetic stand.
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.6. f.        Dispense 900 µl ZymoBIOMICS™ MagWash 2 and mix well by pipette or shaker plate for 1 minute.
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.7. g.       Transfer the deep-well block to a magnetic stand until beads pellet, then aspirate and discard the supernatant. Remove the 96-Well Block from the magnetic stand.
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.8. h.       Repeat the wash (Steps 46.6-46.7).
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.9. i.         Transfer the 96-Well Block onto a heating element (55°C) until beads dry (approximately 30 minutes).
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.10. j.         Heat again at 30°C for 30 minutes
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.11. k.       Dispense 50 μl of ZymoBIOMICS™ DNase/RNase Free Water to each well and re-suspend beads. Mix the beads well for 10 minutes and then transfer the plate onto the magnetic stand for 2-3 minutes until the beads pellet.
SECTION: DNA Extraction 96 Plate (With Eppendorf epMotion) D4302, D4306, D4308
46.12. l.         Transfer the supernatant (containing the eluted DNA) to a clean elution plate or tube. The eluted DNA can be used immediately for molecular based applications or stored ≤ -20ºC for future use.