PCR your DNA Workshop

How do we go from showing the existence of DNA (strawberries, soap and alcohol) to the popular imagery of DNA gels?
This is a consolidated practical go-to version of workshops we have done as Hack-a-Taq and Citizen Science @ Bern. It highlights also the generic lab equipment. You can find similar protocols in other places - the OpenPCR site writes why we may want to PCR some DNA. This is our documentation - we put together the "kit" and "home-made" protocols.

Workflow

With a group of 15-20 people with everyone participating, we see that this workflow can easily take the entire day.
You can stop and continue another time - after the DNA extraction and after the PCR.

1. Get Samples
2. Extract the DNA (PAUSE)
3. Amplify the gene of your interest using PCR (PAUSE)
4. Separate the PCR products by agarose gel electrophoresis
5. Visualize the PCR products (your amplified DNA bits)

What you need to Prepare

First, you need to decide what you will be looking for.
In the Bern Citizen Science Workshop, we decided to take samples from our fellow participants (human cheek epithelial cells) and amplify the serotonin transporter genes to look at the different (Long and Short) forms.
For the Hack-a-Taq weekend, we wanted to see if we had any coliform contamination in the waters, and so we used primers to look for the bacteria.
Designing appropriate primers should be another section - but for many things, there are already primers reported in the literature. You can always start with google searches for PCR primers and the name of your gene of interest. For example, the coliform bacteria primers, we decided upon reading many papers that already checked for its efficacy and specificity.

Basic Materials for all of the steps

• gloves (keep your DNA from mixing with others, handling DNA binding dyes)
• trash bin

Basic Sterilization
If you don't have access to a lab-grade autoclave, use a pressure cooker and let it cook for 20 minutes after the pressure is up. To be official, it has to reach 120oC, 15psi for 15 min. Make sure not to close the bottles all the way (it will explode), and make sure not to fill any bottle with liquids more than 3/4 full (it will overflow).
Fresh packs of many things are sterile enough for these workshops - plastic extrusion methods require heat, and Q-tips are usually irradiated.

Sample Preparation

Materials

• sterile (or new pack) of Q-tips or tooth picks or mini-tongue depressors - (anything you can scratch, and that you can autoclave)
• sterile eppendorf tubes, or equivalent plastic or glass tubes - if you want to concentrate your sample by centrifuging, you need centrifuge compatible tubes
• PCR grade water - or autoclaved de-mineralized/distilled water

Equipment

• Optional Centrifuge to concentrate sample

DNA Extraction

Materials
For bacteria and animal cells:

• Genotyping "Kit" - contains extraction buffer to disrupt the membranes and Proteinase K to digest any proteins bound to DNA. There are many genotyping kits out there - in Bern, we used one of these commercially available kits
  
• - or Make your OWN DNA Extraction Buffer - from Goldenberger et al.:
  • 50mM Tris-HCl pH 8.5 (to buffer the pH)
  • 1mM EDTA (to chelate any divalent cations and prevent DNA digesting enzymes to degrade the DNA)
  • 0.5% SDS (surfactant to burst open the cell and in eukaryotes, nucleus walls)
  • 200ug/ml Proteinase K (to digest any proteins bound to the DNA)

Quick and dirty bursting of cells for bacteria:

• Distilled water

Equipment

• PCR machine or equivalent temperature controlled water bath
• Tube holders

PCR

Materials

• Primer pairs (forward and reverse primers) - these are ordered commercially - find your local supplier. (Microsynth (CH))
  • Human Serotonin Transporter primers
  • Coliform bacteria primers
• PCR mix - commercially available - sometimes they also contain loading buffer for agarose gel loading
• - or make your OWN:
  • Polymerase - Taq is usually sufficient for our purposes unless you want to do some cloning where accurate "copying" is necessary. Also, because pOpenTaq is available opensource - now the initial company seems to have been absorbed by another one.
  • dNTP - these are the building blocks of DNA (deoxy nucleotide triphosphates), usually sold as a mix of the four bases (dATP, dTTP, dGTP, dCTP)
  • 10x PCR buffer (10x concentrated)
    • 100 mM Tris-HCl, pH 8.3
    • 500 mM KCl
    • 15 mM MgCl2
    • 0.01% gelatin (stabilizer, optional)
  • PCR quality water (see above)
  • Finally, mixing all these components requires lost of tips!

Equipment

• PCR tubes - these are smaller and thinner walled than eppendorf tubes for better heat transfer
• PCR machine - or a timer and 3 different temperature water baths
• tube holders
• pipettes (200ul, 10ul sizes)
• Pipette tips for 200ul, 10ul pipettes (DNase, RNase free - i.e. freshly opened is OK)

Agarose Gel Electrophoresis

Materials

• agarose - or agar agar from a store
• TAE buffer - for both the gel and to put into the electrophoresis tank/chamber, here are the final concentrations of the components. Normally, this is prepared as a 50x concentrated solution
  • 40mM Tris HCl
  • 20mM Acetate
  • 1mM EDTA
  • pH 8.3
• Loading buffer - this is just a negatively charged colored dye + glycerol so your samples sink nicely into the wells of the gel
• water
• DNA binding dye, such as SybrSAFE - you can always stain afterwards

Equipment

• Something to boil and melt the agarose
• Pipette and tips (20ul, 100ul or 200ul)
• Electrophoresis System
• tape
• Power source

Gel Visualization

Materials

• DNA binding dye - if you didn't add something already to the gel

Equipment

• Transilluminator - if using SyBRSAFE, Blue LED transilluminator, if using methylene blue, your naked eye will do
• Camera to take the picture of the gel

Step by Step Procedure

Sample Preparation

• To pellet any bacteria in water samples, centrifuge 10 min at 12,000xg

PCR

Basics of PCR can be found explained in many places. The wikipedia PCR page has extensive documentation. If you just want to conceptually visualize what we are doing, here is a video from youtube.

• A typical Taq polymerase PCR mix protocol by New England Biolabs

Agarose Gel Electrophoresis

You don't even need so much agarose - see this experiment using a plastic drinking straw.

References

1. Goldenberger, Perschil, Ritzler and Altwegg, A Simple "Universal" DNA Extraction Procedure Using SDS and Proteinase K Is Compatible with Direct PCR Amplification. Genome Res 1995 pp. 368-70 --- pdf
2. Tan and Yiap DNA, RNA, and Protein Extraction: The Past and The Present Journal of Biomedicine and BiotechnologyVolume 2009 (2009), Article ID 574398, 10 pages
3. Baden et al. Open Labware: 3-D Printing Your Own Lab Equipment PloSBiology (2015) DOI: 10.1371/journal.pbio.1002086