Silage protocols

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Introduction
These protocols are use for the analysis of any forage material, but especially silage.

Making Silage

 * 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)
 * 5. Calculate the amount of water needed to achieve 60% moisture in a 100g Sample.
 * Water (ml)=60-%moisture
 * 6. Weigh out the approproate amount of plant material
 * Amount (g)=100-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, 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.

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)