Silage protocols: Difference between revisions

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:4. Centrifuge filtrate (10-15 ml) at 4,000 g for 10 min.
:4. Centrifuge filtrate (10-15 ml) at 4,000 g for 10 min.
:5. Resuspend pellet in 700 µl TE buffer in a screw-cap microfuge tube.
:5. Resuspend pellet in 700 µl TE buffer in a screw-cap microfuge tube.
:6. Add the following:
:6. Add the following to the tube:
::*500mg glass beads (.1 mm diameter)
::*500mg glass beads (.1 mm diameter)
::*50μl 20% sodium dodecyl sulfate
::*50μl 20% sodium dodecyl sulfate
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:10. Vortex again.
:10. Vortex again.
:11. Centrifuge for 5 minutes at 12,000g.
:11. Centrifuge for 5 minutes at 12,000g.
:12. Extract the Aqueous phase.
:12. Extract as much of the Aqueous phase as you can with a pipette.
::*Your tube should contain four phases now, which starting from the bottom should be: glass beads, Phenol, Cell debris, Aqueous Phase.  
::*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 such up any cell debris.
===Notes===
*If you really want to, after step 4 you can use a soil DNA extraction kit.  These are especaillu useful in removing humic acids that are a common contaminant of corn stover.
Back to [[Richard_Lab:protocols | Protocols]]
Back to [[Richard_Lab:protocols | Protocols]]

Revision as of 17:36, 8 June 2011

Back to Protocols


Introduction

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

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 25g of silage to 25ml of TE Buffer in a small blender.
2. Blend 2X for 10 seconds (each time).
3. Weigh 2g of this mix and add to 8ml DI water (This the first 1:10 dilution of your original sample).
4. Vortex to mix.
5. Perform five more 1:10 serial dilutions by adding 100ul of the previous dilution to 900ul 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 QPCR

1. Add 25g of silage to 25ml of TE Buffer in a small blender.
2. Blend 2X for 10 seconds (each time).
3. Squeeze mixture through four layers of cheese cloth.
4. Centrifuge filtrate (10-15 ml) at 4,000 g for 10 min.
5. Resuspend pellet in 700 µl TE buffer in a 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 (try to keep things as cold as possible).
  • Use a bead beater if you have one.
  • While vortexing it can sometimes be helpful to hold an ice-cube in the same hand as the tube.
8. Place in 60ºC water-bath for 10 minutes.
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.
  • 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 such up any cell debris.

Notes

  • If you really want to, after step 4 you can use a soil DNA extraction kit. These are especaillu useful in removing humic acids that are a common contaminant of corn stover.

Back to Protocols

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