Silage protocols: Difference between revisions
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=Plate Count= | =Plate Count= | ||
1. Add 25g of silage to 25ml of TE Buffer in a small blender | :1. Add 25g of silage to 25ml of TE Buffer in a small blender. | ||
2. Blend 2X for 10 seconds (each time). | :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). | :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. | :4. Vortex to mix. | ||
5. Perform five more 1:10 serial dilutions by adding 100ul of the previous dilution to 900ul water. | :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 (1:10<sup>4</sup>,1:10<sup>5</sup>, 1:10<sup>6</sup>). | ||
6. Plate 100ul of each of these three final dilutions in triplicate | :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. | :7. Calculate the total count per 1g of silage. | ||
::* | ::*For 1:10<sup>4</sup> dilution multiply the plate count by 10<sup>5</sup> to get cfu/g silage. | ||
::* | ::*For 1:10<sup>5</sup> dilution multiply by 10<sup>6</sup>. | ||
::* | ::*For 1:10<sup>6</sup> dilution multiply by 10<sup>7</sup>. | ||
::* | ::*If a plate has more than ~300 colonies,then forget about that plate. | ||
8. Average results | :8. Average results. | ||
=DNA Extraction for QPCR= | =DNA Extraction for QPCR= | ||
Back to [[Richard_Lab:protocols | Protocols]] | Back to [[Richard_Lab:protocols | Protocols]] | ||
Revision as of 17:05, 8 June 2011
Back to Protocols
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
Back to Protocols
