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Project:Plant species testing: Difference between revisions
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→Precipitation extraction method: 17/7/12, 24/7/12
Mycoplasma (talk | contribs) |
Mycoplasma (talk | contribs) |
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* Centrifuge - recover supernatant | * 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) | * 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) | ||
* Add | * Add 100% ethanol, store at -20° overnight (actually for a week) for DNA to precipitate | ||
*We observed white particles on the side of the tube which we assumed to be DNA, but no white strings, which is what we were expecting | *We observed white particles on the side of the tube which we assumed to be DNA, but no white strings, which is what we were expecting | ||
* Centrifuge for 30 mins to make DNA form pellet at bottom of tube. Unclear if pellet had formed, but removed supernatant and resuspended in H2O anyway. | * Centrifuge for 30 mins to make DNA form pellet at bottom of tube. Unclear if pellet had formed, but removed supernatant and resuspended in H2O anyway. | ||
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Conclusion: none of the runs prove we have DNA in the gel, indicating our extractions failed. | Conclusion: none of the runs prove we have DNA in the gel, indicating our extractions failed. | ||
===Repeat without PCR (30/7/12): === | |||
* 2 peas liquified, a few grains of salt and a drop of washing up liquid added | |||
* Incubated at 56°C for 15 mins | |||
* Centrifuge for 5 mins - recover 80 ul of supernatant. | |||
* Add proteinease K (25 ul of 1mg / ml solution to 80 ul of pea template) - incubate at 50°C for 10 mins (action of proteinase k) then 94 C for 10 mins (inactivation of proteinase K) | |||
* Add 70% isopropanol, store at -20° overnight. | |||
