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 Searching for the Ubiquitous Genetically Engineered Machines by (Art)SciencBLR, Hack-a-Taq and Citizen Science @ Bern. It highlights also our Generic Lab Equipment. You can find similar protocols in many 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.

Give some Background

 * Nice explanation series on PCR and agarose gel electrophoresis from Khan Academy
 * A recollection of the invention of PCR by the inventor and Nobel Prize winner Kary Banks Mullis himself.

What is PCR used for?
PCR is a way to make multiple copies of a short segment of DNA. DNA Finger printing - a way to identify individuals based on their DNA Another nice example by (Art)ScienceBLR 2011 iGEM project.
 * Short Tandem Repeat (STR) DNA database and its original publication.
 * DNA Testing: An Introduction For Non-Scientists, An Illustrated Explanation by DONALD E. RILEY, Ph.D.

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) In the meanwhile prepare the agarose gel
 * 5) Separate the PCR products by agarose gel electrophoresis*
 * 6) Visualize the PCR products (your amplified DNA bits)*

* The PCR amplified DNA can have different downstream analyses - by running a PCR, one obtains sufficient DNA for sequencing, to be visualized by gel electrophoresis, or for cloning into a plasmid for further experiments.

Safety and Disposal of Materials
We have conducted these workshops in public spaces. Best practice for safety includes having protective gloves and disposal for some of the DNA binding dyes, such as SYBR Safe (see their FAQ). Article with considerations in choosing the DNA binding dye.

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 Equipment
 * 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
 * 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 like the OpenPCR 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 quality water
 * PCR mix - commercially available - sometimes they also contain loading buffer for agarose gel loading !!! needs a -20C freezer or 4C fridge
 * A practical option may be to get a mix that you can keep at room temperature that already comes in the PCR tubes, with loading buffer included - an example of that here.
 * - 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. !!! needs a -20C freezer
 * dNTP - these are the building blocks of DNA (deoxy nucleotide triphosphates), usually sold as a mix of the four bases (dATP, dTTP, dGTP, dCTP) !!! needs a -20C freezer or 4C fridge
 * 10x PCR buffer (10x concentrated)
 * 100 mM Tris-HCl, pH 8.3
 * 500 mM KCl
 * 15 mM MgCl2
 * 0.01% gelatin (stabilizer, optional)

...there is a reason why most biologists don't make their own mixes...

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 (agarose is a purified form of agar agar from seaweed)
 * 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 + viscosity enhancing compound so your samples sink nicely into the wells of the gel - several loading buffer recipes available in Open Wetware.
 * DNA ladder (for size comparison, from 100-1,000 base pairs works nicely)
 * water
 * DNA binding dye, such as [www.lifetech.com/sybrsafe SYBR Safe] - 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 Equipment
 * DNA binding dye - if you didn't add something already to the gel - Methylene blue is a safer alternative and widely available
 * 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
These procedures have been documented countless of times. There is great documentation out there. Here is our version.
 * Open Wetware DNA protocol

Sample Preparation

 * To pellet any bacteria in water samples, centrifuge 10 min at 12,000xg
 * Check out the (art)ScienceBLR documentation where they extracted DNA from soil samples
 * For (human) cheek epithelial cell extraction
 * take a fresh Q-tip per person (sample specimen!) and pre-wet the tip in clean PCR water
 * scrub the inside of your mouth
 * place in a tube with 200ul of PCR your DNA Workshop buffer
 * CONTROL - control that there are no extra floating DNA in your Q-tip or other reagents by taking the Q-tip, and going through all of the steps of sample preparation without the "sample" - for example, for the cheek epithelial cells, do everything except scurbbing the inside of the mouth.

With Proteinase K

 * 1) Activate the Proteinase K at 75C for 900 sec = 15 min
 * 2) Boil at 95C for 300 sec = 5 min

Simple Osmotic Shock
With bacteria, we can do a quick and dirty osmotic shock protocol. There are protocols where they use more reagents to get cleaner DNA preps, but this one from back in the day just boils them in water.
 * 1) Resuspend your bacterial pellet (from centrifugation) with distilled water (about 300ul)
 * 2) Put in a boiling water bath (or use your PCR machine set to 100C) for 20 min
 * 3) Your bacteria should have popped and released their DNA
 * 4) Optional - you can clean up the prep by centrifuging the debree away 10 min at 12,000xg and taking the supernatant (liquid above the pelleted cell junk).

Fungal DNA Extraction
Still looking for a easy protocol. Generally consists of the following steps: Rapid procedure for the extraction of DNA from fungal spores and mycelia This microwave protocol seems promising: Validation of a Quick PCR Method Suitable for Direct Sequencing: Identification of Fusarium Fungal Species and Chemotypes for Preventive Approaches in Food Safety Food Technology and Biotechnology 52(3) · July 2014.
 * 1) Physically crush the fungal structures with a buffered surfactant solution
 * 2) RNase treatment to remove RNA
 * 3) Partition into aqueous phase using phenol/chloroform/isoamylalcohol extraction
 * 4) Ethanol precipitation

To minimize DNA degradation by enzymes, it is good to proceed to the next step quickly, or pause by putting your samples in the fridge/freezer. DNA can be frozen long term with no major problems.

PCR
Basics of PCR can be found explained in many places. Some examples: If you just want to conceptually visualize what we are doing to the DNA, here is a video from youtube. A typical Taq polymerase PCR mix protocol by New England Biolabs - shows the 2 important parts of the PCR procedure:
 * wikipedia PCR page
 * American Society for Microbiology Protocol
 * Putting together the PCR reaction - your sample DNA and the primers of your choice with the other reagents (PCR your DNA Workshop)
 * First add the water and the primers with the PCR mix - and make bulk - so if there are 20 samples, make 25 times as much required (extra + for the controls so that you don't run out)
 * Making a 25ul reaction (can also do 50ul reactions)
 * Primers are often kept as stocks of higher concentration - as a starting point, use 200nM = 0.2uM final each of forward and reverse primers.
 * Add water so that total of everything brings the volume up to 25ul - this depends on what kind of PCR mix is being used (the freeze dried one vs. commercial 2x PCR mix used, vs home-made)
 * Pipette 24ul of what you mixed in 1 (primer + PCR mix) in the PCR tubes - here you can use the same pipette tip
 * Pipette 1ul of your extracted DNA sample into a tube prepared in 2 - here, if there are 20 samples, use a new pipette tip to make sure not to introduce some contamination of mixes of DNA samples
 * CONTROLS - the control for the PCR reaction
 * 1ul PCR water instead of the extracted DNA sample
 * 1ul Q-tip preparation from the DNA extraction step


 * PCR conditions - this depends on the enzyme (Extension), and also the primers (Annealing)
 * Here is a great guideline page to determine / optimize PCR conditions by NEB. Searching in past DIYBio forum threads may be another way to go.

Agarose Gel Electrophoresis
The wikipedia article is great background reading. Other protocols and descriptions are available.
 * Method Book
 * (Art)ScienceBLR with photos

Gel Casting aka Making the Gel
Agarose gels can be made while the PCR reaction is running.


 * 1) Make the 1x TAE solution
 * 2) In a boilable container, a mini-gel, add 0.5g agarose in 50ml 1x TAE solution (1% agarose), if using agar-agar from the store, use 1g agar-agar in 50ml TAE (2% agar-agar).
 * 3) Cover, heat and stir to dissolve the agarose - microwave OK, but not the food microwave
 * 4) Let it cool (can touch, around 50-60C)
 * 5) You can add the SYBR Safe (1:10,000 dilution = 1 part SYBR Safe to 10,000 parts TAE volume) at this point and swirl gently until uniform (don't make any bubbles!), if using methylene blue not necessary to add here
 * 6) Tape (to water proof) the gel casting tray
 * 7) Pour the agarose gel mix from above
 * 8) Place your sample combs - recommended number of wells = samples + 1 for the DNA ladder
 * 9) Wait til gel hardens

Loading the Gel

 * 1) Take the hardened gel into the electrophoresis tank/system
 * 2) Make sure the sample wells are closer to the negative electrode (DNA is negatively charged, so it will move to the positive electrode!)
 * 3) Pour 1x TAE solution until the gel is covered
 * 4) Add loading buffer into the PCR tubes (if you don't already have loading buffer in your PCR mix)
 * 5) Pipette the samples carefully into the well

Running the Gel
See this experiment in gel preparation using a plastic drinking straw.
 * 1) Double check that the negative electrode is by the loaded DNA samples
 * 2) Hook up the power
 * 3) 100V for a typical mini-gel (5 V/cm) and start the run
 * 4) Look to make sure there are bubbles coming out of the electrodes and the loading buffer color indicators are migrating towards the positive electrode
 * 5) Stop the gel when the color indicator is still on the gel - depends on the PCR product size you want to see

Gel Visualization
Visualizing the amplified DNA fragments requires a dye. This is why SYBR Safe was included in the gel-casting. Otherwise, the gel has to be post-stained, which takes extra time.

The DNA binding dyes stain the DNA, but require excitation by specific wavelengths to be seen. A great comparison of the different dyes, and whether they can be visualized by Blue LED transillumination vs UV transillumination is available by IORodeo. Visualization with Transillumination Methylene Blue Post-staining Methylene blue is considered safer, however is much less sensitive than fluorescent dyes like SYBR Safe. For a robust PCR reaction, this may not be such a big issue.
 * 1) Place the agarose gel onto the transilluminator
 * 2) Wear eye protection
 * 3) Place the filter above the gel to let the excited wavelength through
 * 4) Turn on the blue LED
 * 5) Take a photo
 * 1) Place the agarose gel into a container
 * 2) Add methylene blue 0.02% in 1x TAE so that the gel is covered
 * 3) Cover to prevent evaporation
 * 4) Gently shake at room temperature until bands are visible
 * 5) Take a photo

Primer Pairs From Previous Workshops
human Serotonin Transporter PCR products: the Long form of 419 base pairs (bp), and the Short form of 376 bp.
 * Forward primer: 5’- ATG CCA GCA CCT AAC CCC TAA TGT – 3’, (24)
 * Reverse primer: 5’- GGA CCG CAA GGT GGG CGG GA – 3’. (20)
 * 35 cycles at 98C (30 sec), 68C(30 sec), 72C(60 sec)

Reference: Gelernter et al. Population studies of polymorphisms of the serotonin transporter protein gene. Am J Med Genet. 1999 Feb 5;88(1):61-6. at Citizen Science Bern

Gram negative bacteria tuf gene (house keeping gene) detects both E. coli and Shigella PCR products: 258bp
 * TEcol553: 5′-TGG GAA GCG AAA ATC CTG-3′
 * TEcol754: 5′-CAG TAC AGG TAG ACT TCT G-3′
 * 35 cycles at 98C (30 sec), 58C(30 sec), 72C(60 sec)

Reference: Maheux et al. Analytical comparison of nine PCR primer sets designed to detect the presence of E. coli/Shigella in water samples Water Research 43 (2009) pp. 3019-3028. at Hack-a-Taq

DNA Finger Printing
People have the image of a gel from crime scenes from popular culture - as part of molecular biology / genetics. What is DNA finger printing? history of DNA Fingerprinting - the history as it is told on this site also tells a parallel story how technology gets accepted by society.

Literature

 * 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
 * 4) Tirabassi "How do YOU stain your DNA?" BitesizeBio 2012