Project:Plant species testing

We aim to extract DNA from plants, then PCR out a sequence and run it on a gel 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. If you're trying to repeat any of these and not sure about the procedure, see the links on the front page.

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
Finger 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 dH20.
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:

Electrophoresed for 1 hr on an 100ml 1% agarose gel - 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 DNA extraction and/or 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.

Gel run 10th sept

From right:

1: 250-10000bp ladder (from hackspace). Ladder key
2: Chelex extraction from 17/7 (added 15 minutes after ladder)
3: Isopropanol extraction from 30-31/7
4: Isopropanol extraction from 30-31/7
5: Sample of liquified pea with no other reagents added apart from autoclaved water.

Conclusion: Isopropanol extractions failed because pellet contained too much plant matter - did not filter properly. Liquified pea gives an easily visible smear. Chelex extraction behaved as last time.

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.
EDIT - what happened then...? No evidence on list.