Difference between revisions of "Project:Plant species testing"

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* 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 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.
 
* Add 70% isopropanol, store at -20° overnight.
 +
* Centrifuge at 5000rpm for 40 mins. Isopropanol removed, pellet air dried for 20 mins, then resuspended in autoclaved H2O.
 +
 +
 +
from left in tube holder- pea, ladder, iso
 +
from top in box - ladder - iso - pea

Revision as of 21:09, 31 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 method - 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. Not sure what this is, as genomic DNA should be behind the ladder if not still in the well.

Repeat without PCR: 30/7/12

  • Bashed peas into paste in a glove
  • Placed a 1/2g of sample of template in 250 ul of chelex 5%
  • Incubated at 56°C for 30 mins, with periodic vortexing, the 94°C for 10 mins
  • 10 ul of supernatant visualised on 1% agarose gel, together with a sample containing only loading dye, a sample containing only chelex, and a sample from Will's first chelex extraction of 17/7.

Chelex only and loading dye only samples had no bands - so they are not contaminated with DNA. All pea samples showed a band running ahead of the ladder, becoming more diffuse over time. Conclusion: we think DNAses may be affecting the sample, cutting the DNA into small pieces.

Precipitation extraction method: 17/7/12, 24/7/12

  • 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 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
  • 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.

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.
  • Centrifuge at 5000rpm for 40 mins. Isopropanol removed, pellet air dried for 20 mins, then resuspended in autoclaved H2O.


from left in tube holder- pea, ladder, iso from top in box - ladder - iso - pea