# Silage protocols

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## Introduction

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.

## pH Measurement

1. Weigh out 5g of silage.
2. Add this material to 45ml of nanopure water
3. Put in the shaker for 30 mins.
4. Measure pH by submerging the probe in the liquid.

## Plate Count

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)

### Notes

• 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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