Project:Plant species testing: Difference between revisions
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== First extraction and PCR - 17/7/12 == | == 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 | |||
- 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 | |||
Revision as of 19:52, 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 - 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
