Silage protocols: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 11: | Line 11: | ||
=Plate Count= | =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 (1:10<sup>4</sup>,1:10<sup>5</sup>, 1:10<sup>6</sup>) | ::*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 | |||
*this means you'll use a total of nine plates. | ::*this means you'll use a total of nine plates. | ||
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>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>5</sup> dilution multiply by 10<sup>6</sup> | ||
::*for 1:10<sup>6</sup> dilution multiply by 10<sup>7</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. | ::*if a plate has more than ~300 colonies,then forget about that plate. | ||
8. Average results | |||
=DNA Extraction for QPCR= | |||
Back to [[Richard_Lab:protocols | Protocols]] | Back to [[Richard_Lab:protocols | Protocols]] |
Revision as of 17:03, 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