Difference between revisions of "Project:Plant species testing"
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We used two methods of extraction. Method 1: | We used two methods of extraction. Method 1: | ||
| − | * Blend peas with salt and detergent | + | * Blend peas with salt and detergent according to [http://learn.genetics.utah.edu/content/labs/extraction/howto/ this] protocol. Sieve. |
| − | * Incubate for 15 mins at | + | * Incubate for 15 mins at 60°C (optional step that we didn't take) |
| − | * | + | * Centrifuge - recover supernatant |
| − | * Add proteinease K ( | + | * Add proteinease K (100 ul of 100mg / ml to 300 ul pea template) - incubate for 1 hr at 50C (action of proteinase k) then 94 C for 10 mins (inactivation of proteinase K) |
| − | * | + | * Precipitation to follow |
| − | |||
| − | Method 2: | + | Method 2 (From [http://www.springerlink.com/content/n6293v8808n5g836/ this] paper - we also have full text available): |
| − | * 'Homogenize' with a pestle in 5% chelex solution for 1 min | + | * 'Homogenize' (mush) approx 40 mg pea with a pestle in 200ug of 5% chelex solution for 1 min |
| − | * 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. (Hot plate lost heat in the middle so we boiled for 8 mins) |
* Centrifuge for 1 min - recover supernatant and use as template | * Centrifuge for 1 min - recover supernatant and use as template | ||
| + | |||
| + | PCR: | ||
| + | |||
| + | * Prepare sample with 12.5 ul of Taq ready mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of distilled water. | ||
| + | * Denature at 96°C for 5 mins | ||
| + | * 40 cycles of D = 96°C for one minute, A = 58°C for 30 seconds, E = 72°C for one minute. | ||
| + | * Put paraffin on top after 5 mins (should have been before start) | ||
Revision as of 22:30, 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 according to this protocol. Sieve.
- Incubate for 15 mins at 60°C (optional step that we didn't take)
- Centrifuge - recover supernatant
- Add proteinease K (100 ul of 100mg / ml to 300 ul pea template) - incubate for 1 hr at 50C (action of proteinase k) then 94 C for 10 mins (inactivation of proteinase K)
- Precipitation to follow
Method 2 (From this paper - we also have full text available):
- 'Homogenize' (mush) approx 40 mg pea with a pestle in 200ug of 5% chelex solution for 1 min
- Vortex for 10s then boil for 5 mins, then vortex again for 10s. (Hot plate lost heat in the middle so we boiled for 8 mins)
- Centrifuge for 1 min - recover supernatant and use as template
PCR:
- Prepare sample with 12.5 ul of Taq ready mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of distilled water.
- Denature at 96°C for 5 mins
- 40 cycles of D = 96°C for one minute, A = 58°C for 30 seconds, E = 72°C for one minute.
- Put paraffin on top after 5 mins (should have been before start)