Difference between revisions of "Project:Plant species testing"
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* Blend peas with salt and detergent | * Blend peas with salt and detergent | ||
− | * Incubate for 15 mins at 60 C | + | * Incubate for 15 mins at 60 C (optional step not taken) |
* Filter and centrifuge - recover supernatant | * Filter and centrifuge - recover supernatant | ||
* Add proteinease K (0.8 ul - incubate for 1 hr at 50C then 94 C for 10 mins (inactivation of proteinase K) | * Add proteinease K (0.8 ul - incubate for 1 hr at 50C then 94 C for 10 mins (inactivation of proteinase K) | ||
Line 18: | Line 18: | ||
- Vortex for 10s then boil for 5 mins, then vortex again for 10s | - Vortex for 10s then boil for 5 mins, then vortex again for 10s | ||
- Centrifuge for 1 min - recover supernatant and use as template | - Centrifuge for 1 min - recover supernatant and use as template | ||
+ | |||
+ | Using the PCR machine: | ||
+ | |||
+ | For soak: ( | ||
+ | |||
+ | * | ||
+ | |||
+ | For PCR: | ||
+ | |||
+ | *File 4 enter | ||
+ | *Step | ||
+ | *temp | ||
+ | *time | ||
+ | *temp a | ||
+ | *time | ||
+ | *time e | ||
+ | *Segment 4 - 0 C for 0 seconds | ||
+ | *Cycle count | ||
+ | *Link to stored file - 20 C (to keep |
Revision as of 20:44, 17 July 2012
We are using primers for plants to test and refine our DNA extraction / PCR technique. First project is to test for peas. Inspiration in this paper.
First extraction and PCR - 17/7/12
We used two methods of extraction. Method 1:
- Blend peas with salt and detergent
- Incubate for 15 mins at 60 C (optional step not taken)
- Filter and centrifuge - recover supernatant
- Add proteinease K (0.8 ul - incubate for 1 hr at 50C then 94 C for 10 mins (inactivation of proteinase K)
- Precipitate with rubbing alcohol and meat tenderiser as an enzyme
- Centrifuge and remove supernatant to leave pellet
Method 2:
- 'Homogenize' with a pestle in 5% chelex solution for 1 min - Vortex for 10s then boil for 5 mins, then vortex again for 10s - Centrifuge for 1 min - recover supernatant and use as template
Using the PCR machine:
For soak: (
For PCR:
- File 4 enter
- Step
- temp
- time
- temp a
- time
- time e
- Segment 4 - 0 C for 0 seconds
- Cycle count
- Link to stored file - 20 C (to keep