Automate Assembly Cloning Using a Liquid Handling Platform

Automating Takara In-Fusion® Snap Assembly Cloning on the Eppendorf epMotion® Dennis Condy, Yi-Chien Lu Eppendorf Boris Levitan, Yi Zhao, Michael Haugwitz, Andrew Farmer Takara Bio USA Abstract PCR-based seamless cloning approach is essential to obtain reliable results from scaled-up experiments. On the Eppendorf epMotion 5075t workstation, high accuracy brings a higher value to automation. This application note demonstrates the capability of the epMotion to automate restriction enzyme- and ligation-free cloning using low reaction volumes. This reduces the per-reaction cost without compromising the high accuracy offered by In-Fusion Snap Assembly cloning. Platform: Eppendorf epMotion® 5075t Kit: Takara In-Fusion® Snap Assembly Application: Automated cloning reaction using 96-well and 384-well plates Processing time and tip usage: 96-well plate format: processing 40 samples (10 μL volume) takes 8 min 20 sec (excluding 15 min incubation time) and uses 108 x 10 μL tips. 384-well plate format: processing 40 samples (5 μL volume) takes 14 min 30 sec (excluding 15 min incubation time) and uses 128 x 10 μL tips. Introduction Modern scientific trends lean towards greater convenience and lower reaction volumes while increasing throughput and reproducibility. Many molecular engineering techniques have been adapted to high-throughput platforms, and molecular cloning is no exception. High-throughput cloning methodologies have been developed in the past decade to rapidly clone hundreds to thousands of genes in parallel, especially in the fields requiring high-throughput capabilities such as antibody therapy, proteomics, and synthetic biology. Assay setups in the 384-well format, for example, can be very time-consuming and tedious if done manually, especially when handling very small volumes. Imprecision and pipetting errors caused by the operator can lead to massive variations in the results. The Eppendorf epMotion family of automated pipetting systems is an essential tool for many laboratories looking to achieve consistent results. The epMotion 5075t and other models in the Eppendorf family pipette volumes ranging from 0.2 μL to 1 mL with extreme efficiency and accuracy. PCR-based restriction enzyme-free cloning is a mainstream approach for high-throughput platforms, allowing for inserting any DNA fragment(s) into any vector at any locus without any sequence constraints. In-Fusion seamless cloning allows for restriction enzyme-free, directional, and highly efficient cloning (95%) with extremely low background following a simple protocol. The simplicity of the In-Fusion cloning system makes it ideal for high-throughput workflows without compromising the robustness of cloning technology. Adapting the manual procedure of setting up cloning reactions into liquid handling instrumentations, however, is not always straightforward. In most cases, the manufacturer’s protocol from reagent suppliers is developed based on manual pipetting of a limited number of cloning reactions and does not fully support an automation platform. Here, we established a high throughput cloning workflow using In-Fusion Snap Assembly on the Eppendorf epMotion 5075t to allow scientists easily to adapt a manual procedure to 96-well and 384-well plate formats for an automated platform. APPLICATION NOTE I No. 458 I Page 2 Material and Methods Automation 10 μL In-Fusion Snap Assembly cloning reaction setup > epMotion 5075t > epT.I.P.S.® Motion 10 μL PCR-clean, with filter, sterile > TM10 multi-channel dispensing tool > Eppendorf PCR Sealing Film > MixMate® 10 μL In-Fusion Snap Assembly cloning reaction setup > Thermoadapter for skirted PCR plates, 96- and 384-well > Eppendorf twin.tec® PCR Plate 96 LoBind®, skirted, PCR clean > Eppendorf twin.tec® PCR Plate 384 LoBind®, skirted, PCR clean > 5X In-Fusion Snap Assembly Master Mix > Linearized vector (2.7kb, 53 ng/μL) > epT.I.P.S.® Motion 10 μL, PCR-clean, with filter, sterile > Purified PCR fragment (3.7 kb, 147 ng/μL) > DNAase/RNAase-free distilled water Methods In this experiment, the control vector pUC19 (2.7 kb) was linearized with Hind III restriction enzyme and the insert DNA fragment (3.7 kb) was PCR-amplified with a primer set including 15 bp-sequences homologous to the ends of the linearized pUC19 vector. The primers were designed using the In-Fusion Cloning Primer Design Tool at Takara.com. Two reaction volumes, 10 μL and 5 μL, were tested on a 96well and a 384-well plate, respectively. Within each reaction volume, two automated pipetting approaches were tested for setting up In-Fusion Snap Assembly cloning reactions. The f irst approach was a separate sequential pipetting, in which each reaction component, water, In-Fusion Master Mix, linearized vector, and insert fragments were individually added (“Separate” in Table 1). In the second approach, a reaction master mix of water, In-Fusion Master Mix, and linearized vector (“PreMix” in Table 1) was dispensed into the reaction wells f irst, followed by the transfer of the insert fragment (“PreMix” in Table 1). The experiment was performed on the epMotion 5075t as shown in the worktable setup in Figure 1. Before initiating the procedure, all reagents were dispensed into a skirted 96-well or 384-well PCR plate. The epMotion 5075 was programmed to dispense a series of target volumes (1, 2, 4, and 8 μL). The f irst reagent was delivered with free-jet dispensing, and the following solutions were delivered with a mixing step to ensure no unmixed reagents remain in the tips. Table 1: Set up the In-Fusion Snap Assembly cloning reaction in applications ddH2 O Master Mix (MM) Linearized vector (LV) Premixed H2 O, MM, LV DNA fragment or Ctrl ddH2 O Separate 10 μL 4 μL 2 μL 2 μL 2 μL PreMix 8 μL 2 μL Separate 5 μL 2 μL 1 μL 1 μL 1 μL PreMix 4 μL 1 μLAPPLICATION NOTE I No. 458 I Page 3 Material and Methods Methods (cont.) Forty PCR cloning reactions were performed along with 8 water reactions for negative controls for each condition setup (10 μL-separate, 10 μL-premix, 5 μL-separate, 5 μL-premix). After all the reagents for In-Fusion Snap Assembly cloning were dispensed to the plate, the PCR plate was removed from the epMotion, sealed with Eppendorf PCR fi lm, and mixed at 1200 rpm for 10 seconds on an MixMate®. The PCR plate was then placed back onto the epMotion, and incubated at 50 °C for 15 min, then stored at -20 °C until further analysis. Five wells from the 10 μL reactions and ten wells from the 5 μL reactions were randomly picked from the plates for the transformation procedure (Figure 2). One negative control (no insert) well from each condition was selected (Figure 2). Ten random colonies were chosen from the array of plates corresponding to each reaction and analyzed by Sanger sequencing to determine the cloning accuracy. Sequences were required to be one hundred percent identical to the reference sequence to be counted as accurate. Figure 1: epMotion 5075t worktable for In-Fusion Snap cloning setup Figure 2: Experimental plates, a 96-well (A) and a 384-well (B), setup and sample positions in red for testing cloning effi ciency A. 96-well plate (10 μL) B. 384-well plate (5 μL) Separate PreMix Control Separate PreMix ControlAPPLICATION NOTE I No. 458 I Page 4 Results and Discussion The schematic image (Figure 3) shows an experimental workflow of In-Fusion cloning reaction in a multi-well plate format using epMotion platform. While the entire workflow in the figure can be automated, we applied the automated function only to the In-Fusion enzymatic reaction to focus on this specific reaction step. Information for automating the upstream PCR purification steps using various liquid handling systems is available in separate application notes. We previously confirmed that premixed cloning reaction solution (5X In-Fusion Snap Assembly Master Mix with linearized vector) was stable at room temperature (RT) or 4°C for 2 hours (data not shown). In a high-throughput workflow, linearized vector is often premixed with a cloning reaction master mix and pipetted into each well of multi-well plates ahead of adding unique DNA insert fragments to each well. In this study, we took advantage of the stability of In-Fusion premix solution to test two pipetting approaches. As shown in Table 2, each positive sample yielded 400-1200 colonies after transformation, where >100 colony results represent a successful cloning procedure. All four automated liquid handling conditions showed consistently high efficiency (i.e., colony numbers) and accuracy (i.e., Sanger sequencing; Figure 4). These results indicate that epMotion can handle both 5 μL and 10 μL of In-Fusion Snap Assembly cloning setup and is successful on both separated and premixed pipetting approaches. The sequencing result from a total of 40 positive samples shows >95% cloning accuracy, which indicates utilizing epMotion for automated liquid handling can successfully perform In-Fusion Snap Assembly cloning setup without compromising the high accuracy of the technology. Few colonies in the no insert control are within the normal level of background from un-linearized vectors. Figure 3: At-a-glance image of automated cloning workflow. A target sequence was PCR amplified using forward and reverse primer with 5’ ends homologous to the respective 5’ and 3’ ends of the linearized vector. After purification, the In-Fusion cloning reaction was set up on a 96- or 384-well plate using epMotion 5075t. The construct was transformed into competent cells and plated on selective plates for further analyses. Table 2: Colony count for different pipetting approaches and reation volumes Program Well Colony Count Program Well Colony Count Program Well Colony Count 10 μL Separate A1 448 5 μL Separate A1 1080 5 μL PreMix A13 416 E2 560 A3 816 A15 832 C3 672 C3 680 C15 864 A5 432 E5 768 E17 640 D6 496 G7 832 G19 688 D4 (Ctrl) 2 G9 704 G21 464 10 μL PreMix C7 464 I5 432 I17 480 E8 704 I7 896 I19 848 A9 576 K3 1232 K15 616 C11 496 M1 768 M13 736 A12 544 C11 (Ctrl) 2 C23 (Ctrl) 3 D10 (Ctrl) 0APPLICATION NOTE I No. 458 I Page 5 Results and Discussion (cont.) A. 96-well plate (10 µl) Accuracy: 10/10 700 600 Colony Count (1/100 dilution) 500 400 300 200 100 0 B. 384-well plate (5 µl) Accuracy: 10/10 Accuracy: 10/10 1,200 1,000 800 Accuracy: 9/10* Colony Count (1/100 dilution) Separate Test group No insert control Premix 600 400 200 0 Separate Test group No insert control Premix *10% failure was due to the non-linearized vector. Figure 4: In-Fusion cloning reaction set-up using the epMotion. Two pipetting approaches, separate (labeled as ‘Separate’) and pre-mixed (labeled as ‘Premix’) pipetting, were taken to set up the In-Fusion reaction with the epMotion. Graphed values are the mean colony counts of independent cloning reactions. Error bars show ± standard deviations. Accuracy was determined by Sanger sequencing. Conclusion In this application note, we demonstrate that the highly effi cient restriction enzyme- and ligation-free In-Fusion cloning technology can be implemented on Eppendorf epMotion systems. Furthermore, the epMotion was able to pipette low range volumes with extreme effi ciency and accuracy in each tested pipetting approach, thereby ensuring reproducibility and also reducing per-reaction costs. Product Use Limitations and Safety Information Please read the In-Fusion Snap Assembly manual before performing the method for the fi rst time.