Destination: AWESOME!

Howdy ho budding Biochemologists!

Congratulations! You’re about to embark on a super saver one-way journeyon board the HMS Wonder to the fabulous land of Awesome. En route to the destination, should the conditions permit, we will try to take in the island of Wow and the sacred mountain range of Hooray. To add jeopardy to the excitement, you have all been entered into a special prize draw where the winner will be randomly (seemingly) selected and awarded varying degrees of disaster and disappointment. Some or all of you may not make it to the Promised Land;to those of you who don’t, please don’t take it personally, your failure has been waiting to be revealed since the Big Bang. If you reach those pleasant distant shores will not have travelled in vain, this isn’t like a ridiculouswintry trip to Belgium that you really didn’t think through or something, it’s actually pretty cool. Bon Voyage!

Overview of the process:

  1. DNA extraction
  2. DNA purification
  3. DNA precipitation
  4. PCR
  5. Gel electrophoresis to detect PCR products

Equipment

Micro centrifuge tube and PCR tube (we’ll be mostly using the PCR one, mostly). Fixed volume micropipettes, the one we’ll be using is set to 40ul.

Figure 2 Microfuge tube, left, and PCR tube

  • TE buffer - this stops biological agents from breaking down DNA, which is otherwise extremely chemically stable, which is why it’s used by life to store genes[1]. If buffers were people, this one would be George Cloony, it’s the most famous and is pretty much in anything with DNA in it.
  • Lysis buffer- 1:2 Washing up liquid[2]:TE buffer and a pinch of proteinase K. The helps to break down lipid components to the membranes that separate you from victory (cell membrane and nuclear envelope), the proteinase also helps with this by nonspeficically hydrolysing the membrane proteins, but also seperates the DNA from the nucleosomes and, most importantly, breaks down any DNAses floating about in the mix.
  • Lysis buffer used 06/02/14
  • Lysis buffer version 2: AB LYSIS BUFFER (500ml)

50ml 1M Tris (pH 8.0)

10ml 5M NaCl

50ml 0.5M EDTA (pH 8.0)

100ml 10% SDS

290ml ddH2O[3]

  • Phenol – oily, organic layer that removes proteins (and lipids) which get all up in your business like some kind of jive talking turkey in downstream applications, including the protease which would break down any enzymes like the Taq polymerase essential for PCR
  • Chloroform, another, less harsh, organic solvent removes the phenol
  • Ethanol removes the chloroform
  • Sodium Acetate – High salt solution helps the DNA precipitate out of the ethanol
  • PCR Beads – Contain the Taq DNA polymerase which does the copying of the DNA, MgCl2 needed for Taq and the dNTPs, the building blocks used to extend the DNA
  • Gel loading buffer – makes the final PCR solution sink into the well of the gel for electrophoresis.
  • Agarosegel – a polysaccharide derived from seaweed used at about 2% agarose, TAE buffer and a dash of 20,000x indicator solution (RedSafe)
  • TAE buffer – Contains TE and a spot of sodium acetate to increase the ion concentration to increase the conductivity of the electrolyte used in the electrophoresis.

Step 1: Extracting the DNA

This step gathers the plant material and then releases the DNA from the cells

Figure 3 DNA Density in a plant seed[4]

Figure 4 Anatomy of a Peanut[5]

  1. The highest DNA density in a peanut seed is the embryo, crack open a fresh one and pop out the embryo, get either the leaves or the embryonic root and remember which one you’ve got. Try to crush it with a spatula on a piece of paper.
  2. Now place it in a PCR tube until it’s about a third fullof peanut extractand add 500ul of lysis buffer, and with the plastic tip try to mix it with the buffer as well as possible.
  3. Place it in the 60 oC water bath for 30 minutes.
  4. Break time!

Step 2: Phenol/Chloroform purification

This step removes proteins and lipids

  1. Transfer the plant sample to a microfuge tube and add 500ul of phenol
  2. Mix the contents of the tube until an emulsion forms.
  3. Centrifuge the mixture for 1 minute at room temperature. If the organic and aqueous phases are not well separated, centrifuge again for a longer time.
  4. Use a micropipette to transfer the aqueous phase (top layer) to a fresh PCR tube, but keep all of your old tubes in case things go-pear shaped.
  5. Now add 500ul of phenol again to the aqueous phase, mix, centrifuge it again.
  6. Transfer the top layer to a new PCR tube and add 500ul of chloroform this time, mix, then centrifuge it again.
  7. Remove the aqueous top layer to a new PCR tube, add 500ul of chloroform a second and final time, mix, then centrifuge it again.
  8. Finally remove the aqueous(top) phase again to a new PCR tube then perform the ethanol precipitation
  9. Break Time!

Ethanol Precipitation

Put the rotor buckets for the centrifuge in the freezer, the centrifuge itself in the fridge the night before.

1.Measure the volume of the DNA sample.

2.Add 1/10 volume of 3M sodium acetate, pH 5.2, (final concentration of 0.3 M)- These amounts assume that the DNA is in TE only; if DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration.

3.Mix well.

4.Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition).

5.Mix well.

6.Place on ice or at -20 degrees C for >20 minutes.

7.Spin at maximum speed in a microfuge 10-15 min.

8.Carefully decant supernatant.

9.Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully decant supernatant.

10.Air dry or briefly vacuum dry pellet.

11.Resuspend pellet in the appropriate volume of TE or water.

Step 3 DNA precipitation and resuspension into TE buffer

This step makes the DNA insoluble in ethanol, spins the salt into a pellet and allows you to remove all traces of organic solvents

  1. Spin the cold microfuge tube at full speed for 20 minutes
  2. Carefully pour off as much of the ethanol as you can.
  3. Place in a drying cupboard for 30 minutes
  4. Break Time!

Step 4 PCR (Polymerase Chain Reaction)

This step uses your extracted and purified DNA and makes, hopefull, hundreds of millions of copies of a gene specific to peanuts (the Ara h2 gene[6])

  1. Resuspend the DNA pellet in 100ul of TE buffer
  2. Take 8ul of your DNA solution and add it to a tube containing a PCR bead.
  3. Add 8ul of primer 1
  4. Add 8ul of primer 2
  5. Place in the thermocycler
  6. Finish

Step 5 Agarose gel electrophoresis

This step separates out any DNA that you have and allows you to then visualise your success!

  1. Pour a 3% agarose gel, remember the 20x TAE and the RedSafe DNA stain (has a special fluorescence spectra under UV light when bound to DNA)
  2. Place the gel in 1 litre of TAE buffer so that the buffer covers completely the agarose gel
  3. Add 8ul of loading buffer to your PCR sample
  4. Add 20ul of this solution to a well, run at 120V for 25mins.
  5. Visualise using hand-held UV lamp (or a Transluminator) looking for fluorescent bands.

Figure 5See, it does work sometimes! Check out the barely visible band, this is what it should look like, but much brighter.

Patrick BrannacPage 1 of 7

[1] The “T” is Tris-Cl is a pH buffer set to pH8, to help reduce the activity of DNAses, proteins which break down DNA and are everywhere thanks to various different immune systems which use them to attack viruses which are basically just bits of DNA

The “E” is EDTA, a chelating agent that effectively removes all metal ions in solution, which again the DNAses need in their active sites to cleave the covalent bonds in otherwise stable phosphodiester backbone during hydrolysis.

[2] Contains SDS, sodium dodecyl sulphate, the old skool name of which is sodium lauryl sulfate , is in pretty much all synthetic soaps and detergents these days, even toothpaste.

[3]Lougheed Genetics Laboratory Manual 2005

[4]

[5]

[6]Identification and characterization of a hypoallergenic ortholog of Ara h 2.01. Ramos et al. Plant Mol Biol. 2009 Feb;69(3):325-35