Lum:Microbial Community Analysis

Procedures

Soil Collection

1. Use Soil Core Sampler
2. Collect soil around root zone of the plant
3. Replicates within 1’x1’ area
4. Use soil 5-20 cm from surface
5. Put unused soil in -20°C

Mo Bio UltraClean Soil DNA Isolation Kit with BeatBeater Protocol

1. In Bead Solution (in kit)
   • Add 0.6 g of soil (sieve through 2mm mesh)
   • Add60 µl of Solution S1 then invert several times
   • Add 200 µl of Solution IRS
2. Homogenize in BeadBeater for 10 minutes (2 Cycles of 5 minutes)
3. Centrifuge at 10,000 x g for 30 seconds
4. Transfer solution to a new micro centrifuge tube (expect 400-450 µl)
   • Then add 250 µl of Solution S2
   • Vortex for 5 seconds
5. Incubate at 4°C for 5 minutes
6. Centrifuge for 1 minute at 10,000 x g
7. Avoiding the pellet, transfer all supernatant to a clean micro centrifuge tub3
   • Then add 1.3 ml of solution S3 (careful add, it will reach the brim)
   • Vortex for 5 seconds
8. Load 700 µl onto a spin filter (in kit)
9. Centrifuge for 1 minute at 10,000 x g
10. Discard flow through
11. Repeat 8-10 until all has gone through the filter
12. To spin filter, add 300 µl Solution S4
13. Centrifuge for 30 seconds at 10,000 x g
14. Discard flow through
15. Centrifuge for 1 minute at 10,000 x g
16. Put Spin Filter into a new micro centrifuge tube
    • Add 50 µl of Solution S5 onto white filter
17. Centrifuge for 30 seconds at 10,000 x g
18. Discard Spin Filter
19. DNA is ready!
20. Store in -20°C

Quantification

1. Instument Qubit Fluoromete
2. Setting Broad Range
   • Use Broad Range Standards in BR kit
3. Turn lights off, reagents are light sensitive
4. Prepare Working Solution
   • 199 µl Quant iT Buffer : 1 µl Quant iT Reagent
   • 199:1 for every sample needed (ex.10 samples will be 2000 µl total)
5. Prepare Samples
6. *190 µl working solution + 10 µl standard 1 solution
7. *190 µl working solution + 10 µl standard 2 solution
8. *195 µl working solution + 5 µl of DNA
9. Vortex for 2-3 minutes
10. Incubate 15 minutes
11. Follow On Screen Directions

PCR Notes

1. • Bleach bench
2. *Use filtered tips
   • Make sure all reagents are completely thawed
3. *Spin down tubes before use

Polymerase Chain Reaction (PCR)- Fungal

1. Reaction Mixture per 50 µl reaction
2. *5x buffer : 1x
3. *mgcl2: 2.5 mM
4. *dntps: 0.25mM
5. *3126t: 1.25µM
6. *2234c: 1.25µM
7. *BSA protein: 0.4 µg/µl
8. *Undiluted DNA: 2 µl
9. Hot Start PCR- Add polymerase after denaturation cycle: 0.5 µl
10. Thermal Cycler Conditions
11. *95 °C for 2 minutes
12. *95 °C for 30 seconds
13. *55 °C for 30 Sec
14. *71 °C for 30 seconds
15. *Repeat b-d 29x
    • 71 °C for 5 minutes
16. Expected band size 300-1100 (Ranjard 2000)

Polymerase Chain Reaction (PCR)- Bacterial

1. Reaction Mixture per 50 µl reaction
2. *5x buffer : 1x
3. *mgcl2: 2.5 mM
4. *dntps: 0.25mM
5. *S-D Bact: 1.25µM
6. *L-D Bact: 1.25µM
7. *BSA protein: 0.4 µg/µl
8. *Undiluted DNA: 2 µl
9. Hot Start PCR- Add polymerase after denaturation cycle: 0.5 µl
10. Thermal Cycler Conditions
11. *95 °C for 2 minutes
12. *95 °C for 30 seconds
13. *55 °C for 30 Sec
14. *71 °C for 30 seconds
15. *Repeat b-d 29x
    • 71 °C for 5 minutes
16. Expected band size 450-600 (Ranjard 2000)

pH of Soil

1. In a cup or beaker, mix 40 g of dried and sieved soil with 40 mL of distilled water (or other amount in a 1:1 soil to water ratio) using a spoon or other utensil to transfer the soil.
2. Stir the soil/water mixture with a spoon or other stirrer until it is thoroughly mixed. Stir the soil/water mixture for 30 seconds and then wait for three minutes for a total of five stirring/waiting cycles. Then, allow the mixture to settle until a supernatant (clearer liquid above the settled soil) forms (about 5 minutes).
3. Measure the pH of the supernatant using the pH paper or meter. Dip the pH paper or calibrated pH meter in the supernatant. Record the pH value on the Soil pH Data Sheet. If pH meter requires calibration, gloves should be worn.

Visualize Agrose Gel

1. Place gel into Kodak Imager
2. Turn on instrument
3. Open Kodak Program on computer
4. Click capture
5. Once image has been taken turn off insturment
6. Zoom function on left hand toolbar
7. File-> Save as .bip
8. File->Save Image as .jpeg

Manipulate Photo on photoshop

1. Upload image
2. Image > Adjustment > levels
3. Image > Image size (For Printing)

Making Agrose Gel (Small size BioRad Casting Plate)

1. Use 1000 mL graduated cylinder
2. 35 mL of 5x TBE
3. Fill to 350ml with MilliQ water
4. Mix with parafilm
5. Put 45mL of .5x TBE into 250 or 100mL erlenmeyer flask
6. Add .45g of agose to e. flask
7. Wiegh flask and make note of weight
8. Plug top of flask with kimwipes
9. Microwave until solution is clear (about 45 second)
10. Weigh again and fill to same weight with milliQ water
11. Cool
12. Add 2.25 µl of ethidium bromide
13. Pour into casting plate with comb for wells

Gel Electrophoresis

1. Place polymerized gel into base with wells closest to the black lead
2. Pour .5x TBE into base to the fill line
3. Load Samples
4. Put safety lid on with electrical leads
5. Plug into power supply
6. Turn on power supply and set to 100V
7. About 45 minutes to run half way through gel

Seed Sterilization

1. Scarify lightly with Sand Paper
2. 20 minutes of with 20% bleach
3. 10 minutes sterile water (6x)
4. Put seeds on plate with small amount of water
5. Seal plate with parafilm

Set up Magenta Jars

1. 3 magenta jars and 1 connector needed
2. Stack two majenta jars together
3. *Make a hole on the bottom of the top magenta jar
4. Place cheese cloth at the base of the top magenta jar
5. Fill 2/3 of top magenta jar with 1:1 mixture of vermiculite and sand
6. Place connector and third majenta jar to enclose top
7. Autoclave jars