DNA Extraction

Print Labels for 1.5mL tubes (“Monet, Sample ID, Core Location, DNA")

Measure 200mg of soil in ZR Bashing Bead Lysis Tube, do not exceed 200mg as to not overwhelm the tube

Remove all the lids and add 750uL of ZymoBIOMICS lysis solution

Recap and put in a bead beater for 10 minutes

Centrifuge ZR Bashing Bead Lysis Tube in a microcentrifuge at 16,000 x g for 1 minute

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

Centrifuge at 8,000 x g for one minute (If you get a pellet, pipette supernatant to a fresh tube)

Discard the filter

Add 800uL of Genomic Lysis Buffer and 400uL of ethanol to the filtrate. Mix well

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:)

Centrifuge at 10,000 x g for 1 minute. Discard flow through

Add the rest of the solution from step 9 and repeat step 11

Put the filter in a new collection tube

Add 200uL of DNA pre-wash buffer to the Zymo-Spin IICR Column and centrifuge at 10,000 x g for 1 minute

Add 500uL of g-DNA wash buffer to the Zymo-Spin IICR Column

Centrifuge at 10,000 x g for 1 minute. Discard flow through

Repeat step 16

Get a new collection tube for the filter and change gloves

Add 100uL of elution buffer to the filter and let incubate at room temperature for 1 minute

Centrifuge at 10,000 x g for 1 minute

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.

Put the III-HRC filter into a 1.5mL microcentrifuge tube with cap.

Discard the filter from step 19 and pipette the eluted DNA to the prepared III-HRC filter

The filter may be discarded and the eluted DNA is now in the 1.5mL microcentrifuge tube

Centrifuge at 16,000 x g for 3 minutes

Select dsDNA and follow the prompts from the Nanodrop.

Use 2uL of nuclease free water as the blank before you begin measuring samples

Once the blank has been calibrated use 2uL of sample to measure the concentration and purity

Set up two standards, (Note: keep buffer away from the light as much as possible)

Measure 190uL of buffer and 10uL of standard in Qubit measurement tube

Vortex the tubes briefly

Incubate in the dark for two minutes

Measure 198uL of buffer and 2uL of sample, vortex briefly and incubate in the dark for two minutes

Follow Qubit prompts and measure standards and then sample

Add 200uL of DNA Binding Buffer to the eluted DNA

Transfer the mixture to a Zymo-Spin column in a collection tube. If you have multiple sets of DNA, combine them in this step

Centrifuge for 30 seconds at 16000 x g and discard flow through

Add 200uL of DNA Wash Buffer centrifuge for 30 seconds at 16000 x g then repeat this step again.

Put the filter in a 1.5mL microcentrifuge tube with cap

Add 100uL of DNA Elution Buffer to the filter, let incubate for 1 minute at room temperature

Centrifuge at 16000 x g for 1 minute. Eluted DNA is now ready for use

Print Labels for 1.5mL tubes (“Monet, Sample ID, Core Location, DNA) place on ZR BashingBead Lysis Rack

Measure 200mg of soil in ZR Bashing Bead Lysis Tube, do not exceed 200mg as to not overwhelm the tube

Remove all the lids and add 750uL of ZymoBIOMICS lysis solution

Recap and put in a bead beater for 10 minutes

Centrifuge ZR Bashing Bead Lysis Tube in a microcentrifuge at 16000 x g for 1 minute

Add 600uL of ZymoBIOMICS MagBinding Buffer and 25uL of the beads to 3 96 well plates. Mix well

Add 200uL of supernatant to each 96 well plate so you have 3 sets of the sample

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)

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

j. Heat again at 30°C for 30 minutes

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.

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.

b. Dispense 500 µl of ZymoBIOMICS™ MagBinding Buffer and mix well by pipette

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.

d. Dispense 500 µl of ZymoBIOMICS™ MagWash 1 and mix well by pipette

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.

f. Dispense 900 µl ZymoBIOMICS™ MagWash 2 and mix well by pipette or shaker plate for 1 minute.

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.

h. Repeat the wash (Steps 46.6-46.7).

i. Transfer the 96-Well Block onto a heating element (55°C) until beads dry (approximately 30 minutes).

Centrifuge plates at 3486 x g for 3 minutes before you start

Add 200uL of ChIP DNA Binding Buffer to each volume of DNA sample

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

Repeat step 49 with duplicate samples

Add 200uL of DNA Wash Buffer and centrifuge for 5 minutes at 3486 x g

Centrifuge again to ensure all solution has filtered through to the collection plate

Put Zymo-Spin I-96-XL Plate on an Elution Plate and add 50uL of Elution Buffer

Centrifuge for 5 minutes at 3486 x g to elute the DNA.

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

Add 6mL of BashingBead buffer

Shake on bead beater for ten minutes

Centrifuge at 3486 x g for five minutes

Transfer the supernatant to a clean 50mL tube, there is likely a pellet

Add 18mL of Genomic Lysis Buffer to supernatant and mix well

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

Repeat step 61, because the mixture won’t all fit the first time

Centrifuge at 2000 x g for five minutes once more to ensure the solution has entirely filtered

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)

Add 300uL of DNA Pre-Wash Buffer to the filter and spin at 10000 x g for 1 minute and discard flow through

Add 400uL of g-DNA wash Buffer to the column and centrifuge at 10000 x g for 1 minute, discard flow through

Repeat step 66

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

Transfer the Zymo-Spin III-HRC filter to a 1.5mL microcentrifuge tube

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

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.