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Project:Plant species testing: Difference between revisions
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→Precipitation extraction method: 17/7/12, 24/7/12
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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 ?% ethanol, store at -20° overnight (actually for a week) for DNA to precipitate | ||
* Centrifuge for 30 mins to make DNA form pellet at bottom of tube. Unclear if pellet had formed - in | *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. | |||
*Skipped PCR and ran genomic DNA on a 40mg agarose gel - as with chelex extraction, saw a faint blurry band running ahead of the ladder, and a small amount of stain in the well. | |||
Conclusion: none of the runs prove we have DNA in the gel, indicating our extractions failed. | |||
