Mar 23, 2022
Automated DNA template preparation and quantitation methods
- Jane Romantseva1,
- David Ross1,
- Nina Alperovich2,
- Elizabeth Strychalski2
- 1National Institute of Standard and Technology;
- 2National Institute of Standards and Technology
- DNA template preparation for CFE
Document Citation: Jane Romantseva, David Ross, Nina Alperovich, Elizabeth Strychalski 2022. Automated DNA template preparation and quantitation methods. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkk8rdl5r/v1
Manuscript citation:
Romantseva E.F., Tack D.S., Alperovich N., Ross D., Strychalski E.A. (2022) Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production. In: Karim A.S., Jewett M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007 /978-1-0716-1998-8_1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: March 21, 2022
Last Modified: December 13, 2022
Document Integer ID: 59697
Keywords: cell-free expression, automated DNA extraction, automated DNA purification, DNA template preparation, automated DNA quantitation, automated dna template preparation, methods for dna template preparation, dna template preparation, active dna template, protein production, dna, essential for protein production, reproducibility in cell, automated method, using cell
Disclaimer
Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.
Abstract
Concentrated, intact, and biologically-active DNA templates are essential for protein production using cell-free expression. We present automated methods for DNA template preparation and quantitation towards improving reproducibility in cell-free expression.
Troubleshooting
The following automated methods for DNA template preparation and quantitation were developed for improving reproducibility in cell-free expression and are described in detail elsewhere.
Citation
LINK
These methods were developed using the Hamilton STAR automated liquid handler with the Monitored Multi-Flow Positive Pressure Evaporative Extraction ([MPE]2) module. For questions about methods or implementation, contact Jane Romantseva ([email protected]).
Disclaimer: Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.
1. Load deck with tips for DNA template preparation methods
0_LoadDeckwithTips.hsl 0_LoadDeckwithTips.med 0_LoadDeckwithTips.stp 0_LoadDeckwithTips.sub 2. Aliquot cell culture into tubes and wells
Aliquot pEFR40019 culture.hsl Aliquot pEFR40019 culture.med Aliquot pEFR40019 culture.stp Aliquot pEFR40019 culture.sub 3. Resuspend cell culture for DNA preparation with two kits
Resuspend pEFR40019 culture.prepare for 2 kit DNA extraction.hsl Resuspend pEFR40019 culture.prepare for 2 kit DNA extraction.med Resuspend pEFR40019 culture.prepare for 2 kit DNA extraction.stp Resuspend pEFR40019 culture.prepare for 2 kit DNA extraction.sub 4. DNA extraction using QIAGEN QIAprep 96Turbo kit with MPE2
DNA Extraction.QIAGEN QIAprep 96 Turbo.with MPE2.hsl DNA Extraction.QIAGEN QIAprep 96 Turbo.with MPE2.med DNA Extraction.QIAGEN QIAprep 96 Turbo.with MPE2.stp DNA Extraction.QIAGEN QIAprep 96 Turbo.with MPE2.sub 5. DNA extraction using Omega Biotek Mag-Bind Ultra-Pure kit with MPE2
DNA Extraction.OmegaBiotek Mag-Bind Ultra-Pure.with MPE2.Version2med.hsl DNA Extraction.OmegaBiotek Mag-Bind Ultra-Pure.with MPE2.Version2med.med DNA Extraction.OmegaBiotek Mag-Bind Ultra-Pure.with MPE2.Version2med.stp DNA Extraction.OmegaBiotek Mag-Bind Ultra-Pure.with MPE2.Version2med.sub 6. DNA Purification using Invitrogen PURELink Pro96 kit with MPE2
DNA Purification.InvitrogenPURELink Pro96.with MPE2.hsl DNA Purification.InvitrogenPURELink Pro96.with MPE2.med DNA Purification.InvitrogenPURELink Pro96.with MPE2.stp DNA Purification.InvitrogenPURELink Pro96.with MPE2.sub 7. Load deck with tips for DNA quantitation methods
00_LoadQuantDeckwithTips.hsl 00_LoadQuantDeckwithTips.med 00_LoadQuantDeckwithTips.stp 00_LoadQuantDeckwithTips.sub 8. Aliquot QuantIT Master Mix for DNA quantitation using fluorometry and QuantIT BroadRange kit
DNA Quantitation.Aliquot QuantIT Master Mix.dsDNA Broad Range Assay.hsl DNA Quantitation.Aliquot QuantIT Master Mix.dsDNA Broad Range Assay.med DNA Quantitation.Aliquot QuantIT Master Mix.dsDNA Broad Range Assay.stp DNA Quantitation.Aliquot QuantIT Master Mix.dsDNA Broad Range Assay.sub 9. Prepare DNA samples for DNA quantitation using fluorometry and QuantIT BroadRange kit
DNA Quantitation.Prep Quant Plates.hsl DNA Quantitation.Prep Quant Plates.med DNA Quantitation.Prep Quant Plates.stp DNA Quantitation.Prep Quant Plates.sub Citations
Romantseva EF, Tack DS, Alperovich N, Ross D, Strychalski EA. Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production.
https://doi.org/10.1007/978-1-0716-1998-8_1