Silage protocols: Difference between revisions
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::*While vortexing it can sometimes be helpful to hold an ice-cube in the same hand as the tube. | ::*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. | :8. Place in 60ºC water-bath for 10 minutes. | ||
:9. Place in 95ºC water-bath for 3 minutes | :9. Place in 95ºC water-bath for 3 minutes. | ||
:10. Vortex again. | |||
:11. Centrifuge for 5 minutes at 12,000g. | |||
:12. Extract the Aqueous phase. | |||
::*Your tube should contain four phases now, which starting from the bottom should be: glass beads, Phenol, Cell debris, Aqueous Phase. | |||
Back to [[Richard_Lab:protocols | Protocols]] | Back to [[Richard_Lab:protocols | Protocols]] |
Revision as of 17:27, 8 June 2011
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Introduction
These protocols are use for the analysis of any forage material, but especially silage.
pH Measurement
- 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.
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:
- 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 the Aqueous phase.
- Your tube should contain four phases now, which starting from the bottom should be: glass beads, Phenol, Cell debris, Aqueous Phase.
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