Difference between revisions of "Project:Plant species testing"
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We are using primers for plants 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 primers for plants 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]. | ||
| − | == | + | == 1st attempt - 17 and 18/7/12 == |
| − | + | Extraction (From [http://www.springerlink.com/content/n6293v8808n5g836/ this] paper - we also have full text available): | |
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* 'Homogenize' (mush) approx 40 mg pea with a pestle in 300ug of 5% chelex solution for 1 min | * 'Homogenize' (mush) approx 40 mg pea with a pestle in 300ug of 5% chelex solution for 1 min | ||
| Line 36: | Line 28: | ||
* 100ml of agarose: 1 grams of agarose powder to 100ml of 1% TBE solution | * 100ml of agarose: 1 grams of agarose powder to 100ml of 1% TBE solution | ||
| − | * Microwaved the agarose, cooled to 50 degrees, added | + | * Microwaved the agarose, cooled to 50 degrees, added 5ul of ethidium bromide stock solution |
| + | * Poured agarose into mould and waited to cool | ||
| + | * Ladder = far, template = close | ||
| + | * Electrophoresed - started at 80V, settled at around 130V | ||
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| + | == 2nd attempt == | ||
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| + | * 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 60°C (optional step that we didn't take) | ||
| + | * Centrifuge - recover supernatant | ||
| + | * Add proteinease K (100 ul of 1mg / ml solution to 300 ul of 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 | ||
Revision as of 21:37, 18 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 and primers are in this paper.
1st attempt - 17 and 18/7/12
Extraction (From this paper - we also have full text available):
- 'Homogenize' (mush) approx 40 mg pea with a pestle in 300ug 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)
Electrophoresis:
Quantities of chemicals:
- 1% TBE, 1 part "TBE 10x" to 9 parts water
- Agarose, 1 gram per 100ml of TBE
- Ethidium bromide stock solution, 1 gram of powdered EtBr per 100ml of TBE
- 2ul of ethidium bromide stock solution per 40ml of agarose
Protocol:
- 100ml of agarose: 1 grams of agarose powder to 100ml of 1% TBE solution
- Microwaved the agarose, cooled to 50 degrees, added 5ul of ethidium bromide stock solution
- Poured agarose into mould and waited to cool
- Ladder = far, template = close
- Electrophoresed - started at 80V, settled at around 130V
2nd attempt
- 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 1mg / ml solution to 300 ul of 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