These protocols are use for the analysis of any forage material, but especially silage.
Making Silage (in 50g quantities)
- 1. Weigh out 10g of the material to be ensiled
- 2. Put it in the microwave for 20 seconds, then weigh it again.
- 3. Repeat this last step until the weight stops decreasing.
- 4. Calculate the Percent moisture on a fresh weight basis.
- Percent moisture=100-(10*Final weight)
- This is really a "ball-park" measurement. Accurate measurement should be done using a 105°C oven.
- 5. Calculate the amount of water needed to achieve 60% moisture in a 50g Sample.
- Water (ml)=(60-%moisture)/2
- 6. Weigh out the approproate amount of plant material
- Amount (g)=50-Water (from step 5)
- 7. Place the weighed plant material (from step 6) in your desired sealable container.
- 8. Add the calculated amount of water from step 5.
- If you're adding a silage inoculant, dilute it in this water.
- 9. Seal the container as tightly as possible (vacu-pack if you can).
- 10. Place in an incubator.
- 11. Ensiling should be complete in a couple days, and the silage will be fully matured in a week.
- Measuring the pH is a good way to monitor silage quality, expect a pH below 4.5 for good silage.
- Also, your material should turn somewhat yellow.
- It should not smell rancid (butyric acid), but more like sour milk (lactic acid) or even potentially like vinegar (acetic acid).
- If your bags become extreemely puffy, there's probably a problem; but a little inflation is OK.
Growing Silage Inoculants
- Silage inoculants are grown in MRS medium, and a typical 24 hour culture has 108-109 CFU/ml.
- Inoculant are applied at around 105-106 CFU per dry gram, so one ml of overnight culture is enough to inoculate around 1kg of dry mass.
- Centrifuge the culture and resuspend the cell pellet in the water you are adding to reach your desired moisture content.
- Weigh out 5g of silage.
- Add this material to 45ml of nanopure water
- Put in the shaker for 30 mins.
- Measure pH by submerging the probe in the liquid.
- 1. Add 1g of silage to 49ml of TE Buffer in a 50ml centrifuge tube.
- 2. Vortex thoroughly to mix.
- 3. Weigh 5ml of this mix to 5ml DI water (This represents a 1:100 dilution of your original sample).
- 4. Vortex to mix.
- 5. Perform four 1:10 serial dilutions by adding 1ml of the previous dilution to 9ml water.
- You will use the last three of these dilutions for plating (1:104,1:105, 1:106).
- 6. Plate 100ul of each of these three final dilutions in triplicate.
- This means you'll use a total of nine plates.
- 7. Calculate the total count per 1g of silage.
- For 1:104 dilution multiply the plate count by 105 to get cfu/g silage.
- For 1:105 dilution multiply by 106.
- For 1:106 dilution multiply by 107.
- If a plate has more than ~300 colonies,then forget about that plate.
- 8. Average results.
DNA Extraction for Q-PCR
- 1. Add 10g of silage to 90ml of TE Buffer in a small blender.
- 2. Blend 2X for 10 seconds (each time).
- 3. Filter mixture through a 90μm filter (also known as a #170) and collect 50ml of filtrate.
- 4. Centrifuge filtrate at 4,000 g for 10 min.
- After this step your pellet will look like mud with white specs. I'm told these white specs are lactic-acid-bacteria.
- After this step you can proceed with a soil DNA extraction kit or continue this protocol (see note).
- 5. Resuspend pellet in 700 µl TE buffer in a 2ml screw-cap microfuge tube.
- 6. Add the following to the tube:
- 500mg glass beads (.1 mm diameter)
- 50μl 20% sodium dodecyl sulfate
- 700 μl equilibrated phenol
- 7. Vortex for 3 minutes
- Use a bead beater if you have one.
- Try to keep things as cold as possible.
- 8. Place in 70ºC water-bath for 10 minutes (vortex half-way through).
- 9. Place in 95ºC water-bath for 3 minutes.
- 10. Vortex again.
- 11. Centrifuge for 5 minutes at 12,000g.
- 12. Extract as much of the Aqueous phase as you can with a pipette and place in a new micro centrifuge tube.
- Your tube should contain four phases now, which starting from the bottom should be: glass beads, Phenol, Cell debris, Aqueous Phase.
- Be sure not to suck up any cell debris.
- Be really really sure not to suck up any cell debris.
- 13. Add an equal volume of phenol, centrifuge for 5 min at 12,000g, remove aqueous phase to a new tube (carefully).
- 14. Repeat previous step.
- 15. Repeat previous step except this time use a 1/1 mix of Phenol and Chloroform.
- 16. Add an equal volume of isopropanol
- 17. Put solution in the -80 freezer for 20 minutes (or -20°C for an hour).
- 18. Centrifuge at full speed for 10 minutes.
- 19. Carefully decant the supernatant while leaving the cell pellet in the tube (this pellet is your DNA).
- 20. Leave the tube upside down for 2 minutes.
- 21. Add 200ml of TE buffer and put tube in 70°C water bath for 10 minutes.
- The pellet should dissolve.
- 22. Proceed with DNA purification protocol (likely using a silica column)
- Soil DNA extraction kits are easy to use and are especially useful for removing humic acids that are a common contaminant of DNA from corn stover.
- Humic acids WILL screw up your Q-PCR.
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