Difference between revisions of "Project:Plant species testing"

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* 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)
 
* 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. Unclear if pellet had formed - in future precipitate for longer.
 
* Centrifuge for 30 mins to make DNA form pellet at bottom of tube. Unclear if pellet had formed - in future precipitate for longer.
 +
*PCR and gel to follow.

Revision as of 21:19, 21 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. We are using two methods of extraction. 1st attempt with a chelex protocol, 2nd with alcohol precipitation.

Chelex extraction - 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 300ul 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:

  • 100ml of agarose solution: 1 gram of agarose powder added 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, then covered with 1% TBE.
  • Put ladder into far well, template into near well
  • Electrophoresed for 1 hr - started at 80V, settled at around 130V
  • Visualised under UV light through orange filter - did not see any bands from the template, but ladder was visible, indicating PCR failed.

Repeat without PCR: 21/7/12

  • 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 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.

Precipitation extraction method: 17/7/12, 21/7/12

  • 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)
  • 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. Unclear if pellet had formed - in future precipitate for longer.
  • PCR and gel to follow.