APPLICATION NOTE No. 387Ultimate PCR Optimization with EppendorfMastercycler X50 2D-gradientArora Phang, Tim Schommartz,Eppendorf AG, Hamburg, GermanyAbstractThe new Eppendorf Mastercycler X50 is the only thermalcycler in the market equipped with the innovative 2Dgradient function. PCR optimization, typically of theannealing temperature using gradient function is anestablished technique. Optimization of the denaturationtemperature is less commonly done and typically limitedto applications dealing with complex or GC-richDNA templates.This is mainly due to the high amount of effort requiredto obtain useful optimal result from the combination ofdenaturation and annealing conditions. The 2D-gradientfunction reported herein allows optimization of bothdenaturation and annealing temperatures in just one PCRrun. This provides users with rich amount of informationin the least amount of time and effort, thus greatly shortening the scientific research process.IntroductionSince the inception of PCR, the technique has gone throughnumerous evolution steps. Similarly, the thermal cycler, adevice designed to carry out PCR, has evolved from a simpleheating device to one with numerous functions that allows PCR to be performed more efficiently. Perhaps one ofthe most powerful innovations in the thermal cycler is thegradient function. This function directly targets the fundamental principle of PCR, that the annealing step in PCR isprimer-dependent and the correct temperature for this stepis very ambiguous and hard to predict. Determination of thecorrect annealing temperature generally involves much trialand error and this fine-tuning can be very time-consuming.Thermal cyclers with gradient function are able to simultaneously provide multiple different temperatures at a certainstep. When used at the annealing step, this function canthus reduce the time and effort needed in optimizing theannealing temperature of a primer1, 2.On the other hand, the denaturation step in PCR has lessambiguous working temperature, generally only deviatingslightly from the temperature specified by the manufacturer.This is because most DNA will be completely denaturedat 95 C and most enzymes have a maximum temperaturetolerance around that temperature. However, while not asvariable as primers, each DNA template has its own characteristic and hence a certain degree of variation is unavoidable. Complex DNA or DNA templates with rich GC contentnaturally require higher denaturation temperature. Thus,while PCR might be successful without optimizing the denaturation step, the quality and yield of the PCR might not beoptimal. An optimal PCR is thus to a smaller or larger degreealso affected by the denaturation temperature used2.To date, it is possible to optimize the denaturation andannealing steps of a PCR system by doing two separateruns (by keeping either the denaturation or the annealingtemperature constant while changing the other). To findthe best combination of optimal denaturation and annealing temperatures, one would have to first run a gradient forthe annealing temperature. Subsequently, for each of theannealing temperatures tested, a gradient is then repeatedfor the denaturation step. This would result in multiplePCR runs that is both time- and resource-consuming. Withthe introduction of the new Eppendorf Mastercycler X50however, this difficulty can now be solved. This ApplicationNote will present a new innovative technique called the 2Dgradient that allows for the ultimate PCR optimization withutmost ease and speed.

APPLICATION NOTE I No. 387 I Page 2Materials and MethodsTable 1: PCR condition with two concurrent gradient setting atPCRBio Taq DNA polymerase (NIPPON Genetics) and Hudenaturation and annealing Genomic DNA (Roche ) were used for the followingamplification. PCR reaction master mix containing 1X reacLid105 Ction buffer, 0.25U of enzyme, 0.2 µM of each primer and 20TSP/ESPONng DNA template was prepared. 10 µl of the master mix was Headerdispensed into each respective 96 wells of Eppendorf twin.tec (Eppendorf settings)Lid auto-offONskirted PCR plates. Dispensing was carried out by EppenTemperature modeFastdorf epMotion 5073. Plates were sealed with adhesive PCRInitial Denaturation96 C/5 minfilm and PCR was carried out on Mastercycler X50s.The following primers were used for amplification of thehuman ß-actin gene:Forward primer: 5’- ATCGCCGCGCTCGTCGTC-3’Reverse primer: 5’- TGGGTCATCTTCTCGCGGTTGG-3’Cycling conditions are listed in Table 1. The PCR productswere detected using GelRedTM (Biotium) following agarosegel electrophoresis and visualized using the Gel Doc XR (BioRad ).Cycles: 35xDenaturationGradient at 90-99 C/20 sAnnealingGradient at 52-72 C/20 sElongation72 C/30 sPost-CycleElongationStorage72 C/2 minHold4 C

APPLICATION NOTE I No. 387 I Page 3Results and DiscussionThe new 2D-gradient function of the Mastercycler X50enables optimization of both the denaturation and annealingtemperatures in one PCR run. This was achieved through amatrix-style temperature set-up whereby the first gradient atdenaturation step is set vertically while the second gradientat annealing step is set horizontally. This means that eachof the eight rows of the thermal block has a different temperature at the denaturation step while each of the 12 columnsof the thermal block has a different temperature at the annealing step.Figure 1: 2D-gradient function can be used in a matrix-style optimization of both denaturation and annealing temperaturesconcurrently to find the optimal condition for highest PCR yield.

APPLICATION NOTE I No. 387 I Page 4Hence, for each denaturation temperature (TD), 12 sampleswould be amplified at that temperature (e.g. wells A1–A12would be subjected to 99 C TD while B1–B12 would besubjected to 98.5 C TD). After the denaturation step,samples under the same column would be subjected to thesame annealing temperature (TA ), thus giving rise to 12different TA across the block (e.g. A–H1 would be subjectedto 51.9 C TA and A–H2 would be subjected to 52.3 C TA ).At the end of the completed PCR, the best combination ofdenaturation annealing temperatures can then be determined (Figure 1).Optimal PCR result is defined by maximum yield of thespecific amplicon of interest. Therefore, the aim of PCR isalways first and foremost specificity followed closely byyield. While this can be primarily achieved through optimizing the annealing temperature, there is no guarantee thatthe result obtained is the true “optimal” result. It is alwayspossible that the yield could be increased or the amount ofnon-specific product be reduced.Figure 2 shows the result of the matrix-style optimizationtechnique of the 2D-gradient in amplifying the humanß-actin gene. This PCR system was chosen because of itstemperature sensitive nature. Specific amplification willyield 484 bp fragments while sub-optimal condition will giverise to non-specific amplification visible as a 350 bp artefactin the gel.Ordinarily, gradient optimization is only performed for theannealing step at a fixed denaturation temperature at ca.95 C. Taking the example from Figure 2, when 95.6 C isused, gradient result for annealing step showed that 65.9 Cgives the best yield with small amount of non-specific product and at 70.5 C, only specific product will be obtained.Depending on the objective of the PCR, both of these temperatures can be considered “optimal” conditions that areusually sufficient for most applications.However, in certain cases such as low target copy number,a small difference in yield can be crucial to the application.In the example above, it can be clearly seen that 95.6 C isnot an optimal TD for this PCR system. By lowering the TDto 93.4 C, the specific bands almost doubled in intensity.In addition, the results in this study showed that increasing TD leads to decreasing non-specific amplification. ForPCR systems where non-specific amplification is a problem,especially those with multiple bands, running a gradient atdenaturation step would be especially beneficial. Hence the2D gradient allows users to easily obtain a rich amount ofinformation about the characteristic of their PCR system,which in turn is beneficial for various application objectivessuch as increasing yield or resolving non-specific amplification problems.ConclusionThe 2D-gradient function of the Mastercycler X50 allowsusers to simultaneously optimize both denaturation andannealing temperatures to determine the conditions forcombined optimal yield and specificity for best PCR result.Not only does the convenience of this function allow usersto save much time and effort in their optimization work, italso has important implications for applications relating tolow target copy number and GC-rich targets. In addition,this function is highly useful in troubleshooting non-specificamplification issues.Figure 2: PCR optimization of ß-actin gene with 2D gradienttechnique.

APPLICATION NOTE I No. 387 I Page 5References[1] Ong, W.K. (2010) Using the gradient technology of the Mastercycler pro to generate a single universal PCR protocolfor multiple primer sets. Eppendorf Application Note 220.[2] Gerke, N. (2013) Straightforward PCR optimization and highly flexible operation on the dual block thermocyclerMastercycler nexus GX2. Eppendorf Application Note 289.Ordering informationMastercycler X50sMastercycler X50aMastercycler X50pMastercycler X50hMastercycler X50I*Mastercycler X50r*Mastercycler X50t*Mastercycler X50i*Order no. International6311 000.0106313 000.0186315 000.0156316 000.0196303 000.0106305 000.0176306 000.0106301 000.012Order no. North hernet cable, 5 m6313 070.0086313070008* To operate this unit, it needs to be connected to a Mastercycler X50 s,a,p, or h. Up to 9 units can be connected to a Mastercycler X50 s,a,p, or h.Your local distributor: AG · 22339 Hamburg · Barkhausenweg 1 · [email protected] · www.eppendorf.comwww.eppendorf.comRoche is a registered trademark of F. Hoffmann-La Roche AG, Swiss. Bio-Rad is a registered trademark of Bio-Rad Laboratories, Inc., USA. GelRedTM is a registered trademark of Biotium, Inc., USA.Eppendorf , the Eppendorf Brand Design, epMotion , Eppendorf twin.tec and Mastercycler are registered trademarks of Eppendorf AG, Germany.All rights reserved, including graphics and images. Copyright 2017 by Eppendorf AG.

Eppendorf AG, Hamburg, Germany APPLICATION NOTE No. 387 Abstract The new Eppendorf Mastercycler X50 is the only thermal cycler in the market equipped with the innovative 2D- gradient function. PCR optimization, typically of the annealing temperature using gradient function is a