Streamlined emulsion PCR protocol
by Tatjana Schütze and Jörn Glökler, Labtimes 01/2011
Current protocols for emulsion PCR require the addition of diethyl ether and tedious sonifiction and precipitation steps. Jörn Glöklers group has simplified the process to match routine as well as high throughput applications.
Emulsion PCR (ePCR) is a powerful PCR method that has demonstrated its usefulness in many applications, such as reduction of amplification bias, cloning for next generation sequencing, enhanced multiplexing and haplotyping. The available protocols, however, are not amenable to high throughput experiments, such as the amplification step in SELEX (Systematic Evolution of Ligands by EXponential Enrichment) experiments. We thus adapted the emulsion protocol to work without any complicated equipment like tissue lyser, sonifier, or custom-made stirrers that needed decontamination to be recycled.
Making extensive use of emulsion PCR: Jörn Glökler (in the middle) and his co-workers Hannsjörg Braun, Julia Repkow, Tatjana Schütze and Nicole Greiner (from left to right).
Instead, a normal vortexer found in almost every lab is employed to do the emulsification step. The tubes can either be fixed by an adhesive tape or put into a fitting tube attachment, if available. Since we tried to avoid an ePCR, in which the biotinylated product is captured on expensive magnetic beads, we thought of a simple way to break up the emulsion and purify the product. Earlier publications employed highly volatile and explosive solvents, such as diethyl ether, followed by nasty precipitation steps, in which precious pellets may be lost due to Murphy’s Law. We recognised that butanol was quite good at breaking up the emulsion, but trace amounts of the oxidised product butyric acid would have made social life more challenging in our lab! Fortunately, isobutanol did the same trick without the unfavourable side-product.
Next, we combined the resulting mixture with conventional spin-column PCR clean up kits. Fortunately, most of them are compatible with our isobutanol approach and yield a clean PCR product ready for subsequent experiments. Briefly, for a typical 50μl aqueous PCR sample a 300μl oil phase is put together from 73% Tegosoft DEC, 20% mineral oil, and 7% ABIL WE. The oil phase should be made as a fresh stock and chilled on ice before addition to the aqueous phase. A standard Vortex-Genie 2 vortexer can be used to emulsify the sample at full speed for 3-5 minutes, preferably in a cold room. Most PCR cyclers have a maximum sample volume of 100μl, so the sample may have to be split into three aliquots. The cycling reaction itself can be performed according to standard programmes (our approach has recently been published in more detail in Schütze et al., Anal. Biochem. 2011 March 1; 410 (1):155-7).
The aliquots are pooled and the emulsion is broken by the addition of 1ml isobutanol, followed by brief manual vortexing. The amplified DNA can be purified from this mix by most commercially available PCR cleanup kits. In this case the binding buffer is added according to the respective manual and the mixed phases are separated again in a centrifuge. The resulting aqueous and interphase containing the DNA is put on the spin column for purification. This may call for sequential loading steps due to the increased volume. The remaining washing and elution steps are done according to the manufacturer’s manual. Note that each micelle has a limited maximum yield of DNA product. We observed that 15 PCR cycles are sufficient to amplify a single molecule to reach this limit. If no product is visible after purification due to little initial occupancy of micelles, a subsequent ePCR can be performed right on top.
By applying our KISS (keep it simple and stupid) protocol, ePCR can be performed in most molecular biology labs without acquiring additional hardware and meticulous handling. This will enable scientists to quickly evaluate whether ePCR can enhance or even enable some of the more challenging PCR experiments. For those who like to have everything readily assembled, a commercial kit based on our protocol is already available.
Last Changed: 10.11.2012