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Project:Plant species testing: Difference between revisions
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==== Repeat without PCR: 21/7/12 ==== | ==== Repeat without PCR: 21/7/12 ==== | ||
*Extraction: same procedure, but started with approx 50 mg pea | *Extraction: same procedure, but started with approx 50 mg of pea in 600ul chelex. (This is much more reactant than in the paper and a waste of chelex, but our scale only goes down to 100mg. Would be good to get a smaller one.) | ||
*Electrophoresis: same procedure but used 0.5% instead of 1% agarose solution. | *Electrophoresis: same procedure but used 0.5% instead of 1% agarose solution as we are running genomic DNA not PCR product. | ||
*Saw a faint, blurry band, running ahead of the ladder, which we assumed to be the genomic DNA we were looking for. | *Saw a faint, blurry band, running ahead of the ladder, which we assumed to be the genomic DNA we were looking for. | ||
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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 70% isopropanol, wait 10 mins for DNA to precipitate. (We should have done this for longer, possibly in the freezer) | * Add 70% isopropanol, wait 10 mins for DNA to precipitate. Small amount of DNA visible in isopropanol (We should have done this for longer, possibly in the freezer, to get more DNA) | ||
* Centrifuge for 30 mins to make DNA form pellet at bottom of tube. | * Centrifuge for 30 mins to make DNA form pellet at bottom of tube. Unclear if pellet had formed - in future precipitate for longer. | ||
