Acoustic Liquid Handling Enables 40x Reduction in Volume for DNA Assemblies
Written by Ken A on March 13, 2019
Miniaturization throughout a workflow is a key part of lowering costs and speeding up the synthesis of DNA constructs. Here, we'll take a closer look at this process and see how using the NEB NEBuilder® HiFI DNA Assembly Cloning Kit and the Labcyte® Echo® 525 Liquid Handler can achieve miniaturization by forty-fold while successfully assembling a construct over five modular pieces. Sequencing verification using the MiSeq® Nextera XT protocol also reduced costs by limiting reagent volumes. Lowering cost while increasing throughput to produce high-quality synthetic DNA constructs is a win-win combination that will allow scientists to investigate a broader biological landscape.
To demonstrate the miniaturization results, we used phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), a gene that is commonly studied in human cancer research as a real-world example. PIK3CA was divided into four modular pieces based on domains of interest. Of these four pieces, three have known cancer-linked mutations (R38H, E542K, E545K, and H1047R). The construct was engineered for future transfection into mammalian cells, and those prerequisites, along with bacterial replicative machinery constituted the 5th modular piece. These modular DNA assemblies were next transformed into bacterial cells. These transformants were isolated using the Echo® 525 Liquid Handler to array 8 columns of 25 nL per construct transformation onto OmniTrays. The results of using this miniaturization process were successful and validated by qPCR and NGS.
The use of modular DNA pieces in a construction pipeline offers lower cost and faster screening results. The Echo® 525 Liquid Handler along with the NEBuilder HiFi kit adds to these advantages by allowing a 500 nL reaction volume down from the standard 20 μL (a forty-fold reduction). In turn, this means only 1.25 fmoles of input DNA was needed compared to 500 fmoles that was typically recommended. This dramatic scale-down has other advantages across the process chain. The Echo® 525 Liquid Handler, using small volumes, saves operator time as well as the incubator space through the OmniTray plating. In addition, using acoustics enables the transfer small volumes (25 nL) of cell suspension with precision, thus eliminating typical problems associated with tip-based systems on poured agar plates. Using colony qPCR on the Echo® 525 Liquid Handler, false positives from background colonies that grow despite antibiotic selection can be removed before isolating plasmids to substantially boost efficiency. This approach correctly predicted 100% of vector background and 74% of the insertion deletion constructs from the 192 samples that were tested. Finally, the advantages of miniaturization extend to sequencing steps. Using a miniaturized Nextera XT protocol with the Illumina MiSeq, reaction volumes can be reliably decreased by ten-fold.
In summary, with the Labcyte Eco Acoustic Technology, you can now achieve what was previously impossible. Achieving a forty-fold reduction in volume for complex, modular DNA assemblies using the NEBuilder HiFi kit integrated with a Labcyte Echo 525 Liquid Handler reduces cost while boosting throughput levels.
Read the full application note here.
For more info on Echo liquid handling and synthetic biology, go to www.labcyte.com/synbio.
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