TITLE: Protocol for DNA Extraction and Quantitative PCR Detection of Verticillium dahliae from Soil
AUTHORS: David Wheeler, Jeness Scott, Jeremiah Kam Sung Dung, Dennis Allen Johnson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the extraction of DNA from relatively large volumes of soil and subsequent real-time quantitative PCR (qPCR). The qPCR protocol is a modification of the protocol presented by : </div><div class = "text-block">Wei, F., Fan, R., Dong, H.-T., Shang, W.-J., Xu, X.-M., Zhu, H.-Q., Yang, J.-R., and Hu, X.-P. 2015. Threshold microsclerotial inoculum for cotton Verticillium wilt determined through wet-sieving and real-time quantitative PCR. Phytopathology 105:220-229.</div></div>

[STEPS]
?. [Pre-Extraction Sample Preparation:]
The soil cores are combined, air-dried and processed through a sieve (No. 10/2.0 mM). Dried soil samples are stored in a cool dark place until use. When stored in the fridge, keep them in sealed plastic bags to prevent the soil from absorbing moisture.
?. [Pre-Extraction Sample Preparation:]
Place a 5-10 g subsample of soil into a 15 or 50 ml conical polyethylene tube. Cover the tube with a KymWipe to allow moisture and air to move through the sample and keep the sample in the tube. Freeze the subsample of soil at -80°C for at least 3 hours.
?. [Pre-Extraction Sample Preparation:]
The frozen subsample is then freeze dried under a vacuum using a lyophilizer until dry. Drying time varies and ranges from 4 to 24 hours, but could be longer depending soil type.
?. [DNA Extraction]
DNA Extraction Kit: MP FastDNA™ SPIN Kit for Soil (MP Biomedicals; cat no.: 116560200; cost is $230-290 for 50 reactions or 25 soil samples). 100 ml of 100% ethanol (not included in the kit) needs to be added to the concentrated SEW-M solution prior to use.Twice as much lysing matrix E (MP Biomedicals; cat no.: 116914050) is used as compared to other kit components.
?. [DNA Extraction]
Add 500 mg of freeze-dried soil to a lysing matrix E tube (supplied with the kit).a.	Use two tubes per soil sample so that DNA is extracted from a total of 1 gram of soil per sample.
?. [DNA Extraction]
Pre-grind the samples in a FastPrep-24 5G (MP Biomedicals; cat no.: 116005500 ) for 40 sec at a speed setting of 6.0.
?. [DNA Extraction]
Add 828 µl of sodium phosphate buffer (supplied with the kit) to each tube.
?. [DNA Extraction]
Add 150 µl of nonfat powdered milk suspension (133 mg/ml) to each tube (this is not supplied with the kit).
?. [DNA Extraction]
Add 122 µl of MT buffer (supplied with the kit) to each tube.
?. [DNA Extraction]
Homogenize in the FastPrep machine for 40 seconds at a speed setting of 6.
?. [DNA Extraction]
Centrifuge at 14,000 X g for 10 min.
?. [DNA Extraction]
Transfer supernatant to a clean 2.0 ml microfuge tubes (these tubes are not supplied with the kit).
?. [DNA Extraction]
Add 250 µl PPS (Protein Precipitation Solution, supplied with the kit) and mix by inverting the tube 10 times.
?. [DNA Extraction]
Centrifuge at 14,000 X g for 5 minutes to pellet the precipitate.a.	When transferring the supernatant to the 1 ml tube containing binding matrix (steps 11-12) avoid transferring any of the precipitate.
?. [DNA Extraction]
Shake the binding matrix suspension to re-suspend and add 1.0 ml to each 15 ml tube.a.	The two 0.5 g subsamples will be pooled in step 12, so only one 15 ml tube for each soil sample is needed.
?. [DNA Extraction]
Add the supernatants from step 10 to the 15 ml tube containing binding matrix.
?. [DNA Extraction]
Invert by hand for 2 min to allow the DNA to bind to the binding matrix. Place the tubes in a rack for 5 minutes to allow the silica matrix to settle in the tube.
?. [DNA Extraction]
Remove and discard 0.75 ml of the supernatant by pipetting. Be very careful not to disturb the silica matrix.
?. [DNA Extraction]
Gently re-suspend the binding matrix in the remaining amount of supernatant by pipetting.
?. [DNA Extraction]
Transfer 600 µl of the mixture to a spin filter and centrifuge for 1 min at 14,000 X g.
?. [DNA Extraction]
Empty the catch tube and add 500 µl more of the re-suspended silica matrix mixture. Repeat steps 15 and 16 until all of the silica matrix mixture has been added to the spin filter.
?. [DNA Extraction]
Add 500 µl of prepared SEWS-M and gently re-suspend the pellet using the force of the liquid from the pipet tip.a.	Ensure that the ethanol has been added to the SEWS-M before use.
?. [DNA Extraction]
Centrifuge at 14,000 X g for 1 minute. Empty the catch tube.
?. [DNA Extraction]
Add 500 µl of prepared SEWS-M.a.	It is not necessary to re-suspend the pellet during this rinse.
?. [DNA Extraction]
Centrifuge at 14,000 X g for 1 minute. Empty catch tube.
?. [DNA Extraction]
Without the addition of any liquid, centrifuge again at 14,000 X g for 2 minutes. This additional spin removes any excess alcohol that might be present. Discard the catch tube and place the spin filter in a clean catch tube (provided in the kit).
?. [DNA Extraction]
Air-dry the spin filter for 5 minutes at room temperature to allow residual ethanol to evaporate.
?. [DNA Extraction]
Gently re-suspend the binding matrix in 100 µL DES. (DES is the molecular-grade water that is provided with the kit). Incubate at 55°C for 5 min in a heat block.
?. [DNA Extraction]
Centrifuge at 14,000 X g for 1 min.
?. [Removal of PCR Inhibitors]
Post-DNA Isolation Inhibitor Removal: OneStep™ PCR Inhibitor Removal Kit (Zymo Research; cat no.: D6030; $102 for 50 reactions or 50 soil samples).
?. [Removal of PCR Inhibitors]
The columns are prepped according to the manufacturer’s instructions: 1) snap off the base; 2) remove the green cap; 3) insert into a collection tube; and 4) spin in a microcentrifuge at exactly 8,000 X g for 3 min.a.	If the HRC matrix is dry add 400-600 µl water prior to prepping the column.
?. [Removal of PCR Inhibitors]
Move the prepped column to a 1.5 ml centrifuge tube.
?. [Removal of PCR Inhibitors]
Add the DNA suspension from step 25 to the prepped sample column.
?. [Removal of PCR Inhibitors]
Centrifuge at 8,000 X g for 1 min.
?. [Removal of PCR Inhibitors]
Store the DNA in the freezer (-20°C).
?. [Quantitative PCR (adapted from Phytopathology 105:220-229)]
PCR cocktail: All samples are analyzed in triplicate (a total of three 20 µl reactions for each soil sample). Data are averaged across the three reactions.  ABCD1Reagent[Initial]Volume/
  20
  µl reaction[Final]2ABI
  SYBR Select MasterMix2X10 µl1X3Forward
  Primer: Vd-F929-94710 µM2 µl1 µM4Reverse
  Primer: Vd-R1076-109410 µM2 µl1 µM5Molecular-grade
  waterNA4 µlNA6DNA sampleNA2
  µlNA
ABCD1Reagent[Initial]Volume/
  20
  µl reaction[Final]2ABI
  SYBR Select MasterMix2X10 µl1X3Forward
  Primer: Vd-F929-94710 µM2 µl1 µM4Reverse
  Primer: Vd-R1076-109410 µM2 µl1 µM5Molecular-grade
  waterNA4 µlNA6DNA sampleNA2
  µlNA
?. [Quantitative PCR (adapted from Phytopathology 105:220-229)]
Standard Curve: A standard curve should be included with every qPCR run. Extract V. dahliae genomic DNA from mycelia or conidia grown in pure culture. Quantify DNA with the Qubit Fluorometer (ThermoFisher Scientific; cat no.:Q33226) according to the manufacturer’s instructions.    Another fluorometric quantification method can be used, but UV-absorbance-based quantification should not be used. Prepare a ten-fold serial dilution of DNA to use as the template for the standard curve.. The DNA concentration should range from 0.5 ng DNA/µl to 0.5 fg DNA/µl A no template (water) control should also be included with every qPCR run. Prepare a ten-fold serial dilution of DNA to use as the template for the standard curve.. The DNA concentration should range from 0.5 ng DNA/µl to 0.5 fg DNA/µl A no template (water) control should also be included with every qPCR run.
?. [Quantitative PCR (adapted from Phytopathology 105:220-229)]
Thermal Cycler Conditions (Applied Biosystems StepOnePlus Real-time PCR System; cat no.:4376600) 10 min @ 95°C 40 cycles of 30 s @ 95°C, 30 s @ 60°C, and 30 s @ 72°C (measure data).  Melt curve analysis from 60°C, gradually increasing 0.3°C/s to 95°C.