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Project:Plant species testing: Difference between revisions
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Project:Plant species testing (view source)
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We | We aim to extract DNA from plants, then PCR out a sequence and run it on a gel to test and refine our DNA extraction / PCR technique. First project is to test for peas. Inspiration and primers are in this [http://www.epochlifescience.com/Product/SpinColumn/EconoSpin%20Plant%20Seed%20gDNA.pdf paper]. We are using two methods of extraction. 1st attempt with a chelex protocol, 2nd with alcohol precipitation. | ||
== Chelex extraction method - 17 and 18/7/12 == | == Chelex extraction method - 17 and 18/7/12 == | ||
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* 'Homogenize' (mush) approx 40 mg pea with a pestle in 300ul of 5% chelex solution for 1 min | * 'Homogenize' (mush) approx 40 mg pea with a pestle in 300ul of 5% chelex solution for 1 min | ||
* | * Finger 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: | 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 | * Prepare sample with 12.5 ul of Taq ready mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of dH20. | ||
* Denature at 96°C for 5 mins | * 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. | * 40 cycles of D = 96°C for one minute, A = 58°C for 30 seconds, E = 72°C for one minute. | ||
