EDTA buffer is a chelating agent that inhibits enzymatic degradation of DNA and RNA in a solution.
EDTA buffer is an agent that steals ions from molecular machines that need it to make DNA or RNA in a solution go bad.

What is it for?

Enzymes that modify, degrade and synthesize DNA and RNA usually require magnesium ions. EDTA buffer inhibits such metal-dependent enzymes by sequestering metal ions (primarily magnesium and calcium) from the solution. Thus EDTA buffer is a widely used component in buffers and solutions where there are biological products you wish to maintain the integrity of and/or reactions you may wish to suppress.

EDTA buffer is a component of TE Buffer (Tris/EDTA), a protective storage medium for DNA and RNA, and: TAE Buffer (Tris/Acetic Acid/EDTA) and TBE Buffer (Tris/Boric Acid/EDTA), both used for gel electrophoresis. EDTA buffer is also used as an anticoagulant for the storage of blood and preventing clumping of cells in liquid suspension.

Why do you say “buffer”?

Strictly speaking (as a pretend chemist for less than 24 hours), I thought a buffer was a solution designed to maintain a particular pH, whereas this buffer’s primary purpose is to sequester magnesium to suppress enzymes involved in degrading DNA. I find the term buffer a little confusing in this regard and feel chelator would be a better term. However, I’m horrendously unqualified and all the literature seems to refer to this solution as an “EDTA buffer”, but I just wanted to register my discontent¹.

What do I need?

Reagents

• EDTA disodium salt² (FW 372.24)
• Sodium hydroxide (FW 40) pellets
• Milli-Q water (at least the volume of solution you wish to make)

Equipment

• A suitable weighing scale (an analytical scale is preferred, but its maximum weight may be too low and you’ll require a top pan balance instead)
• Realtime pH probe
• Magnetic stirrer and flea

Glassware

The volume of your required glassware will depend on the volume of EDTA buffer you wish to produce. The values in brackets are the containers suggested for a recipe to produce 0.5M 200ml.

• A volumetric flask capable of holding the exact target volume of your recipe (200ml)
• A beaker capable of holding about 50-75% of the target volume of your recipe (250ml)
• A bottle (Duran Flask) capable of holding the target volume of your recipe (250ml)
• A bottle (Duran Flask) to store a suitable volume of MQ water (if not already available)

Bits and pieces³

• Lab spatulas
• Foil
• Measuring cylinder (that can contain the volume of solution you wish to make)
• Funnel
• Access to an autoclave, and autoclave tape
• Patience

What are those things?

EDTA

Ethylenediaminetetraacetic acid (widely referred to as EDTA because nobody can spell or say ethylenediaminetetraacetic) is a chelating agent, capable of sequestering metal ions (including calcium, iron and magnesium). The molecule is hexadentate (“six-toothed”): a cool word describing its claw-shaped structure that is capable of binding very strongly and very effectively to an atom in six places.

This feature of EDTA makes it rather ubiquitous and useful in many industries. For example, EDTA softens water to allow ingredients of soaps, shampoos and laundry detergents to work more efficiently. EDTA is also used to preserve and stabilise cosmetics, eye drops and skin care products in the presence of air, prevent discolouration of dyed fabrics in the production of textiles and can be used as a preservative for food (especially to prevent oxidative decolouration). EDTA can be used to treat many instances of heavy metal poisoning (lead, mercury and others) via chelation therapy, binding to heavy metals in the blood for safe excretion through urine. EDTA can also chelate excess iron from the blood, which can reduce the complications of blood transfusions. EDTA is used extensively in the analysis of blood, primarily as an anticoagulant.

EDTA is an essential medicine according to the World Health Organisation.

Sodium hydroxide

Sodium hydroxide (familiarly named caustic soda) is a commonly used alkali with wide industrial applications including; pulping wood in the production of paper, refinement of bauxite ore to aluminium oxide for the production of aluminium, and the manufacture of soaps and detergents. Sodium hydroxide is also widely used in the preparation of foods, including chemical removal of skins from fruit and vegetables, processing of cocoa, poultry and soft drinks. According to Wikipedia, the unique crust of German pretzels and flavour of Chinese noodles are down to their preparation in sodium carbonate and lye-water respectively.

Sodium hydroxide is highly effective as an industrial cleaning agent, capable of dissolving grease, oils and fats. It is a common component of strong oven cleaner, glass and steel degreasers and drain openers – capable of hydrolysing hard to break down proteins in hair.

It is often used in the laboratory to as a means to raise the pH of solutions.

It is also used by serial killers to dissolve bodies⁴.

Milli-Q Water

Distilled, deionised and filtered water. Ion mass spectrometers will have trouble picking up more than a few parts per million. Really, just, really fucking pure water.

pH probe

An instrument for measuring the pH of a solution by measuring electric potential between two electrodes: the glass and reference electrode. Free ions in the solution (or lack thereof) cause a differential in charge across the inner and outer surfaces of a glass bulb which contains metal salts and surrounds the glass electrode. As the pH on the inside of the glass bulb (that surrounds the glass electrode) is known, pH can be quantified by measuring the differential in conductivity between the inner (glass) and outer (reference) surfaces of the glass membrane.

It is important to calibrate the pH meter before every use (or at at the start of the day, if it is to be used) to ensure accurate readings, as the calibration of the glass electrode will drift through use and time. Calibration should be done with at least two buffers (at either side of the scale of interest). Fancier models will also note the temperature during calibration to correct for variation in pH caused by temperature during actual use later. After use, ensure to follow instructions on cleaning the probe as the electrodes must be kept free of contamination. Typically after use a probe will be rinsed with deionised water, blotted dry and returned to its storage buffer (some form of neutral buffer that does not encourage ions to diffuse out of the electrode).

Solutions like EDTA buffer are only as good as the pH meter that they are buffered with, so it is important this instrument is well cared for. Ensure to follow product guidance for correct cleaning and storage of pH probes in both short and long-term, as these parameters vary between model and manufacturer.

Volumetric flask

A piece of glassware calibrated to contain a precise volume (at a particular temperature), used for precise dilutions and measures of stock solutions. I asked my supervisor why we have volumetric flasks when glass beakers and bottles are typically graduated with volume markings:

“Are they not that accurate then?”
“Awh jesus christ you may as well be measuring everything with your eyes closed!”

How do I make EDTA buffer happen?

Calculate recipe mass

The volume and concentration of EDTA buffer you want to make will depend on what you intend to use it for. For me, I was preparing an EDTA buffer as a component of TAE (Tris/Acetic Acid/EDTA) buffer for gel electrophoresis. A 50x TAE recipe requires 100ml of EDTA buffer at a concentration of 0.5M; a 10x TAE buffer will thus require 20ml. I settled for 200ml as a reasonable volume of EDTA buffer to make; not so much that it will sit in the lab for the next decade⁵, and not so little that the rather laborious effort will need to be repeated any time soon.

To determine the mass of EDTA required for the recipe, we work out how many moles of EDTA should be dissolved in our buffer to obtain the desired concentration (0.5M) in a given volume (200ml). You can adjust the equation below for your own recipe by altering the desired volume and concentration:

A 200ml solution of EDTA with a concentration of 0.5M will contain 0.1 moles. We can now derive the number of physical grams of EDTA that will yield this many moles using its molar mass⁶ as printed on the label⁷:

If you house a distrust for equations, we can also confirm this value with some empirical thinking given the molar mass of EDTA. Considering the molar mass of EDTA is equal to the number of grams required to make a 1 mole per litre (1M) solution, we can derive that the mass for half the concentration (0.5M) will be half the molar mass: 186.12g. For our recipe however, we do not wish to produce a litre, but 200ml. To maintain the desired 0.5 moles per litre concentration in a smaller volume (200ml) we require just a fifth (1L / 200ml = 5) of the already halved mass: independently verifying our value of 37.224g. Mathematics works! It’s important to note that 37.224g will produce a 0.1 moles per 200ml solution, which is another way of expressing 0.5 moles per litre, or 0.5M.

Pre-prep

• Translate hieroglyphs on the Milli-Q water dispenser and dispense Milli-Q water into a Duran flask

Prepare the EDTA solution

• Turn on your scale, wait until it is ready (it may beep, or just show zeroes)
• Place your beaker on the scale, wait for the value to stabilise and tare the scale

• Carefully spatula out the required amount of EDTA for your recipe and realise how futile attempting to measure out a mass correct to four decimal places is

• Move the beaker off, or out of your scales, and turn off the scale, transport the beaker to your magnetic stirrer

• Add between half and three quarters of your target volume of Milli-Q water (e.g. ~150ml for our 200ml recipe) to the EDTA

• Place your beaker on a magnetic stirrer, slip the “flea” into the solution, turn on the magnet and fetch your pH meter

Buffer the solution to pH 8.0

• Remove your pH meter from its storage buffer and calibrate it if necessary (see above)

• With your solution still stirring, insert the pH meter probe and wait for it to stabilise
• Begin adding sodium hydroxide pellets one by one; each time, wait for the pH to plateau and stabilise before adding another

• As you add more sodium hydroxide pellets, the pH should slowly wave towards pH 8.0, at which point almost all of the EDTA should have dissolved
• Once you have finally stabilised the solution as close to pH 8.0 as you dare, leave the stirrer and pH meter on for a few more minutes to verify your work
• If you are sure and happy with the pH, turn off the pH meter, clean the probe (as per instructions) and return to its storage buffer solution
• Turn off the stirrer, retrieve the flea, rinse, and return it to the flea storage box before it gets misplaced

Accurately raise to target volume

• Pour the contents of the beaker into the volumetric flask
• Rinse the beaker with a small volume of Milli-Q water, swirl and add this to the volumetric flask (repeat until the volumetric flask is filled to the graduation mark)

Sterilise

• Transfer the contents of the volumetric flask to a suitably sized Duran flask
• Give the Duran flask a suitable label (0.5M EDTA Buffer, your name, lab, today’s date, etc.)
• Add a strip of autoclave indicator tape
• Loosen the lid of the flask and sterilise the solution in the autoclave

What do I do now?

• Tighten the lid only once the solution has cooled after autoclaving (to prevent the flask shattering)
• Store at room temperature
• Open and pour inside a laminar flow hood only, it’s a sterile product now!
• Put a picture of the bottle on Twitter, well done, you are a god damn alchemist now

How do I fuck it up?

• Waste time using a scale whose maximum weight limit is exceeded by your container and EDTA
• Impatiently dump all your sodium hydroxide pellets into the EDTA+Milli-Q solution, ruin the buffer by overshooting pH 8.0 and have to start over
• Use a poorly maintained or uncalibrated pH meter: the pH is probably the most important property of this product!
• Blow up your hard work by leaving the lid on tight and exposing it to multiple atmospheres in the autoclave

PCR is a laboratory protocol for generating significant numbers of copies of a subsequence of a DNA template, via repeated exposure to an enzyme capable of synthesizing molecules of DNA.

How does it work?

• Short nucleotide sequences called primers are specifically made-to-order to bind (complement) to the start and end of a sequence of interest (for some extracted DNA)
• DNA (the template) is prepared with those primers, dNTPs (basically loose nucleotides) and a polymerase enzyme
• Prepped solutions of between 20-50ul are inserted in a thermal cycler: a machine that alters the temperature to control an exponential DNA amplification via repeated cycles (typically around 30) consisting of three stages:
  • Denaturing template DNA melted at high temperature to yield single strands (exposing strands to primers)
  • Annealing temperature lowered¹ to anneal primers to single stranded template
  • Extending² polymerase binds to both ends of primers and begins to elongate a new strand with the floating dNTPs
• Each cycle creates two new double stranded DNAs from each molecule of DNA present, theoretically the DNA product doubles with each cycle³
• Typically can amplify subsequences of template up to 10kbp

What do I need?

Reagents

• Ice box containing:
  • Taq polymerase
  • Taq buffer
  • Template DNA
  • dNTP mix
  • Forward and reverse primers
• Fridge box containing:
  • HPLC-grade water

Equipment

• Vortex
• Centrifuge
• Thermal Cycler
• Access to HPLC-grade water dispenser and UV Crosslinker

Bits and pieces⁴

• P2, P10, P100, P200, P1000 pipettes (and appropriate tips)
• Eppendorf tubes (500ul, 1.5ml)
• PCR tubes (200ul) + caps (if not attached)
• Multi-format tube rack
• Waste bin
• Gloves

What are those things?

Taq polymerase

A highly thermostable polymerase enzyme (a molecular machine for assembling long chains of nucleic acids) isolated from (and named after) the Thermus aquaticus bacterium; an extremophile that is capable of thriving in high temperature environments (favouring 70°C, but tolerating anything between 60-80°C). Polymerase drives the elongation or extension process of PCR. In the late 1980s, it was discovered that polymerase isolated from Thermus aquaticus could actually withstand the temperatures involved in the annealing step where DNA is melted into its two strands. The polymerase was refined and mass produced for commercial sale; now PCR could be completed without re-adding a polymerase at the end of every cycle!

Taq buffer

PCR buffers attempt to maintain optimal conditions for the activity of polymerases during PCR. Various ingredients can chelate ions that are required for enzymatic activity to reduce degradation of reagents, and unwanted reactions.

Template DNA

Your already extracted and purified DNA sample that contains some sequence that you desire to amplify.

dNTP Mix

Named so as deoxynucleoside triphosphate doesn’t roll off the tongue so well. dNTP mix is essentially a grab bag of the four nucleotides. During the elongation cycle of PCR, polymerases utilize free dNTPs to synthesize new chains of nucleic acids to create complementing strands.

Primers

A pair of short sequences (15-30bp) of nucleic acids designed to complement two ends of a target subsequence of interest on your template DNA. Good primers are 40-60% GC-content, have similar annealing temperatures and should not be self-complementary, or complementary to another primer in the mix.

HPLC-grade water

High-performance liquid chromatography (HPLC) is a technique to identify and separate individual components of a mixture. HPLC-grade water is deionized, filtered, UV-filtered and in general, pretty fucking clean. The goal is to prevent contamination of reagents with nucleases.

How do I make the PCR happen?

Pre-prep

• Gather equipment, ensure your reagents are not depleted, check whether someone has stolen the power lead for the thermal cycler
• Place tube racks in freezer to keep them cold (this helps maintain the integrity of reagents)
• Collect HPLC water (if necessary) and run through UV crosslinker to denature any residual proteins

Prep

• Retrieve tube racks from freezer
• Move dNTPs, primers and template from ice box onto tube rack to thaw, it is essential that these are returned to the ice box as soon as possible once fully thawed

NEVER allow Taq Polymerase to reach room temperature.

Ensure reagents have fully thawed to avoid aspirating solutes of incorrect concentrations.

• Briefly vortex and centrifuge (a few seconds at ~5-10Krpm) Taq buffer and dNTP mix

The buffer must be vortexed to ensure its components are mixed thoroughly.

Prepare a working dNTP mix (if required)

dNTP mix is often shipped at a high concentration (100mM) and in such cases must be diluted to a more practical “working mix” before it is practical to pipette into PCR tubes. This also prevents having to repeatedly freeze-thaw your master mix.

• Calculate the volume of master dNTP mix required to create a more practical solution; say 250ul at a concentration of 2mM:
  
• Aspirate and dispense the solvent first (it is easier to pipette a small volume into a larger one). For our 250ul working mix that contains 5ul of the master mix, we must dispsense 245ul of HPLC water into a 1.5ml Eppendorf tube.
• Vortex and centrifuge the dNTP mix briefly if you have not already done so
• Add 5ul of master mix to the new working mix tube
• Aspirate and dispense repeatedly and carefully to wash the pipette tip and mix the new solution
• Return the 100mM master mix and new suitably labelled 2mM working mix to the ice box

Preparing the PCR tubes

• Lay out the necessary number of required PCR tubes on a cold rack
• Calculate all necessary dilutions before you begin pipetting (consider your protocol parameters; reaction size, desired dilutions of template, primer and dNTP mix):
  • dNTPs
    
  • Primers (forward and reverse)
    
    Although the protocol specification requires a final concentration of between 0.1-1.0uM of each primer, it seems that in general (your mileage will vary), people tend to add excess to give a final concentration up to 2uM. For example 2ul of a 50uM (50pmoles/ul) working primer solution.
  • Template
    
• Remove Taq Buffer from ice box and pipette the volume required by your protocol (my protocol stated 10ul) into all tubes, return the temperature-sensitive Taq Buffer to ice (or the freezer) as soon as possible

It is highly recommended that Taq Buffer is the reagent to be added first. As a buffer, it is responsible for preventing unwanted enzymatic activity such as denaturing (or early annealing) of template DNA and primers.

• The rest of the reagents can be added in no particular order, with the exception of Taq Polymerase, which comes later:
  • dNTPs
  • Primers (ensure both forward and reverse primers are added)
  • Template DNA
• For each tube, sum the volumes of its reagents (don’t forget the polymerase, which is not yet in the tube) and subtract this total from the target volume required by your protocol (again, here, 50ul)
• Add those amounts of HPLC water (the volume to add may differ between tubes if differing volumes of primer or template were added) to bring up the total volume of each sample tube to the target volume (less the polymerase)
• Ready (switch on and program) the thermal cycler before adding Taq Polymerase (reactions begin as soon as it is added, albeit at room temperature)
• Remove Taq Polymerase from ice, vortex gently and centrifuge briefly to remove excess from the walls of its tube⁵
• Add between 0.5–2.0 units of polymerase per 50ul reaction, our protocol recommended 1.25u, I added slightly more to make it easier to aspirate with a pipette, return to ice (or freezer) as soon as possible
• Seal PCR tubes (close lids or seal caps⁶)

Thermal Cycling

• Load tubes into thermal cycler immediately (your reaction has begun!)

If you are feeling particularly prepared, you could preheat the lid of your cycler to ensure the hot-start PCR begins more quickly.

• Ensure lid is as tight as possible (if it has a lid that needs manual tightening to push the heated block⁷ against the tops of tubes)
• Load and check program schedule (does it have a sensible run time? Has someone in your lab accidentally sabotaged it in the last five minutes?)

Ensure there is an infinite store step at less than 5°C following the end of the final cycle of your program. Unless you want all of your work destroyed at room temperature. This is especially important if you are running PCR before going home for 12 hours.

• Run program!
• Watch in horror as you allow the machines to take over everything and probably ruin your experiment

What do I do now?

• PCR product must be kept in the fridge or freezer
• Verify fragments of the expected size (or anything at all) were amplified with gel electrophoresis

How do I fuck it up?

There are a multitude of ways that PCR can fail. Due to the number of reagents required in each tube, and lots of pipetting it is quite trivial to make a mistake. Helpfully, it is typically not possible to establish the cause and the process must be repeated. Lots of attention to detail is required, especially if there is more than one template, or more than one set of primers that make up individual reactions.

• The easiest way to fuck up PCR is to forget something, each PCR tube needs all of the components and even the smallest distraction can cause you to skip, forget or duplicate a step
• Applying the wrong template or primer to a particular reaction tube
• Fucking up your mathematics (usually by forgetting to ensure all the molarity or volume units are the same) for dilutions, yielding low concentration of a reagent (in general it seems PCR is more prone to failure under conditions where reagents are in concentrations that are too low, rather than too high — with the exception of buffer⁸).
• Contaminate your reagents by forgetting to change your pipette tip, this is particularly bad as it destroys your expensive reagent to boot
• Suboptimal annealing temperatures were used during thermal cycling (consult the documentation for your primer set)
• Your template DNA is damaged (poor storage, bad preparation)
• Your primers suck (or are damaged, or perhaps one of the pair binds to another location)
• You might have forgotten to vortex your Taq buffer before use and it happened to have separated during storage
• Adding polymerase first might have caused the reaction to start too early
• Water was not sterile
• The day ends in a ‘y’