Project:Plant species testing

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.
• 23ul of sample + 2ul of loading dye/glycerol mix electrophoresed at 80V for 30 mins. No staining observed.

Conclusion: Extraction inadequate again. (Probable cause was that sample contained plant matter, hence pellet was this not DNA. We also ran liquified pea alone with no other reagents other than autoclaved water - observed a smear.

Email and photos here.

Repeat without PCR (1/8/12):

• 5.3 grams of crushed pea added to 20ml of distilled, sterile water and a shake of salt. Solid removed and 3ml of detergent added
• Shake of meat tenderiser added to break down proteins
• Centrifuged and supernatant removed - solids discarded.
• 5ml of 70% added to 10ml of template for precipitation
• Interface removed and as much liquid as possible removed from this
• Pellet dried at 60°C in thermocycler.

(in each ladder tube there is 14 ul)

1- empty 2- ladder 3 - pea (diffused)

6 - chelexed pea (diffused) 8 - ladder