The Monarch Plasmid Miniprep Kit is a rapid and reliable method for the purification of high quality plasmid DNA. This method employs standard cell resuspension, alkaline lysis, and neutralization steps, with the additional benefit of color indicators at certain steps to easily monitor completion. Unique wash buffers ensure salts, proteins, RNA and other cellular components are removed, allowing low-volume elution of concentrated, highly pure DNA. Protocols are fast and user friendly, saving you valuable time. Elution in as little as 30 μl provides concentrated DNA for use in downstream applications, such as restriction digests, DNA sequencing, PCR and other enzymatic manipulations. Designed with sustainability in mind, Monarch kits use significantly less plastic and responsibly-sourced, recyclable packaging.
Prevent buffer retention and salt carry-over with optimized column design
Reduce hands on time with faster protocols and less spin time
Monitor completion of certain steps using colored buffer system
No need to add RNase before starting
Easily label columns using tab and frosted surfaces
Buffers and columns available separately
Significantly less plastic used when compared with other kits
Responsibly-sourced and recyclable packaging
No hazardous materials fees
Specifications
Culture Volume:
1-5 ml, not to exceed 15 OD units
Binding Capacity:
up to 20 μg
Plasmid Size:
up to 25 kb
Typical Recovery:
up to 20 μg. Yield depends on plasmid copy number, host
strain, culture volume, and growth conditions.
Elution Volume:
≥ 30 μl
Purity:
A260/280 and A260/230 ≥ 1.8
Protocol Time:
10½ minutes of spin and incubation time
Compatible
Downstream
Applications:
restriction digestion and other enzymatic manipulations,
transformation, transfection, DNA sequencing, PCR, labeling,
cell-free protein synthesis, etc.
Monarch Plasmid Miniprep Kit Protocol Monarch Plasmid Miniprep Kits consistently produce more concentrated plasmid DNA with equivalent yield, purity and functionality as compared to the leading supplier
Preps were performed according to recommended protocols using 1.5 ml aliquots of the same overnight culture. One microliter of each prep was digested with HindIII-HF (NEB #R3104) to linearize the vector and the digests were resolved on a 1% w/v agarose gel. Plasmid DNA purified using the Monarch Plasmid Miniprep Kit produces transfection efficiencies equivalent to or better than plasmid DNA purified using the Qiagen QIAprep® Spin Miniprep Kit
Plasmid DNA encoding constitutively expressed GFP (pEGFP-C2) was prepared using either Monarch Plasmid Miniprep Kit or Qiagen QIAprep Spin Miniprep Kit. Four different cell lines (Cos-7, HEK293, HeLa, and Huh-7) were grown to 80-90% confluence and transfected with 100 ng of each plasmid, in complex with 0.3 μl Lipofectamine 2000, and 10 μl Opti-MEM. Five replicates for each cell type were performed using both DNA preps. GFP expressing cells were counted by flow cytometry 48 hrs post-transfection with a minimum of 2000 events collected per well. Average percentage of cells expressing GFP from all replicates is graphed and used as a measure of transfection efficiency. DNA from Monarch Plasmid Miniprep Kit is reproducibly compatible with DNA sequencing.
Plasmid DNA from three separate preps was sequenced using BigDye® Terminator chemistry on an Applied Biosystems 3730XL DNA Analyzer. The electropherograms demonstrate the quality of the DNA is reproducible.
Kit Components
The following reagents are supplied with this product:
Prevent buffer retention and salt carry-over with optimized column design
Reduce hands on time with faster protocols and less spin time
Monitor completion of certain steps using colored buffer system
No need to add RNase before starting
Easily label columns using tab and frosted surfaces
Buffers and columns available separately
Significantly less plastic used when compared with other kits
Responsibly-sourced and recyclable packaging
No hazardous materials fees
Properties and Usage
Storage Temperature
25°C
Troubleshooting
No DNA Purified
Reagents added incorrectly. Check protocol to ensure buffers were added in the correct order and that the sample is bound, washed and eluted in the correct sequence.
Plasmid lost during growth of culture. Ensure proper antibiotic is used at correct concentration in order to maintain selection during growth. Do not sub-culture amplicillin-maintained cultures to avoid depletion of antibiotic by secreted β-lactamase. Use a fresh plate and avoid selecting satellite colonies when innoculating the culture.
Low DNA Yield
Incomplete lysis. Ensure cell pellet is completely resuspended before addition of Plasmid Lysis Buffer (B2) and that color changes from light pink to dark pink. Take care to not use too many cells. If culture volume used is larger than recommended, scale-up buffers B1-B3 to ensure proper processing of the sample.
Plasmid loss during growth, see previous section.
Low-copy plasmid selected. Increase amount of cells processed and scale buffers accordingly.
Lysis of cells during growth. Harvest culture during transition from logarithmic growth to stationary phase (typically 12–16 hrs for growth of cultures in LB) to avoid lysis of cells common during extended periods of cell growth.
Incomplete neutralization. The sample tube should be inverted a sufficient number of times to produce a complete color change to yellow. Cell debris will appear in abundance. Nothing should be floating on the surface after centrifugation, but rather should be compacted into a pellet.
Incomplete elution. Larger elution volumes and longer incubation times can increase yield of DNA off the column, at the cost of dilution of the sample and increased processing times. For typical plasmids in the 3–10 kb range, the recommended elution volumes and incubation times are sufficient. For the purification of larger plasmids, heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield.
Low DNA Quality
Plasmid is degraded. Some E. coli strains (HB101 and the JM series) have high levels of endogenous endonuclease. Avoid using these when possible. If these strains are used, keep samples on ice during prep and ensure the Plasmid Wash Buffer 1 step is performed.
Plasmid is denatured. Using Plasmid Lysis Buffer (B2) introduces sodium hydroxide to the DNA. Extended incubation in the presence of sodium hydroxide can separate the strands, or denature the plasmid. Adhere to the protocol and ensure this step is completed within 2 minutes and promptly move on to the neutralization step.
Plasmid is contaminated with genomic DNA. Vigorous mixing after cell lysis and before pelleting of cell debris may cause shearing of the host cell chromosomal DNA and should be avoided. Additions of Plasmid Lysis Buffer (B2) and Plasmid Neutralization Buffer (B3) should be followed by careful inversion mixing. Do not vortex.
Improper storage. Ensure DNA is eluted in DNA Elution Buffer or nuclease-free water to maintain integrity and store at –20°C. DNA should not be stored in solutions containing magnesium.
Low DNA Performance
Ethanol has been carried-over. Ensure final wash spin time is 1 minute to enable complete removal of the wash buffer from the column. Use care when transferring column to a new tube for the elution step to ensure column tip does not contact column flow-through. If there is any doubt, re-spin the column for 1 minute.
Excessive salt in sample. Ensure both Plasmid Wash buffers have been utilized according to the protocol. Unlike other commercial kits, all wash steps in the Monarch Plasmid DNA Miniprep Kit protocol are required. All steps should be performed as described to ensure the recovery of high-quality plasmid DNA.
Excessive carbohydrate carried over. Similar to the endogenous nucleases, strains like HB101 and the JM series, have high amounts of endogenous carbohydrate that can interfere with downstream enzymatic manipulations of plasmid DNA. Be sure to follow the protocol and make sure the Plasmid Wash Buffer 1 step is included.
View the Troubleshooting Guide for DNA Cleanup and Plasmid Purification
The kit should be stored at room temperature. Always keep buffer bottles tightly closed and keep columns sealed in the enclosed zip-lock bag. After Plasmid Neutralization Buffer (B3) is opened, it should be stored at 4°C. For information regarding the composition of buffers, please consult the Safety Data Sheets. Proper laboratory safety practices should be employed, including the use of lab coats, gloves, and eye protection.
Monarch Plasmid Miniprep Kit protocol
Learn how to do a plasmid miniprep using the Monarch Plasmid Miniprep Kit.
Tips for using the Monarch Plasmid Miniprep Kit
Optimize your plasmid miniprep results with these quick tips for using the Monarch Plasmid Miniprep Kit.
How to recycle your Monarch Kit components
Learn how you can easily recycle all of the components in your Monarch Kits.
Protocols & Manuals
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].)
Back to basics: Important things to keep in mind when purifying plasmids and DNA fragments
Dos and Don'ts of Plasmid Minipreps
Usage Guidelines for the Monarch Plasmid Miniprep Kit (#T1010) When Working with Low Copy Plasmids
Troubleshooting Guides
Troubleshooting Guide for DNA Cleanup and Plasmid Purification
Publications
Vladimir Potapov and Jennifer L. Ong*
(2017)
. Examining Sources of Error in PCR by Single-Molecule Sequencing PLOS.;
PubMedID: 5218489
Fomenkov, A., Vincze, T., Mersha, F., Roberts, R.J.
(2018)
. Complete genome sequence and methylome analysis of Bacillus caldolyticus NEB414. Genome Announcements.;
6 (6), e01605-17.. PubMedID: 29439055 , DOI: 10.1128/genomeA.01605-17.
Vladimir Potapov, Jennifer L. Ong.
(2017)
. Examining Sources of Error in PCR by Single-Molecule Sequencing. PLOS One.;
PubMedID: 28683110
Citations
Kanpiengjai A, Nguyen TH, Haltrich D, Khanongnuch C.
(2017)
Expression and comparative characterization of complete and C-terminally truncated forms of saccharifying a-amylase from Lactobacillus plantarum S21. International Journal of Biological Macromolecules;
PubMedID: 28587961
Luck A, Yuan X2, Voronin D, Slatko B, Hamza I, Foster JM4.
(2016)
Heme acquisition in the parasitic filarial nematode Brugia malayi FASEB;
PubMedID: 27363426
Wei, H., Yan, B., Gagneur, J., Conradt, B.
(2017)
Caenorhabditis elegans CES-1 snail represses Pig-1 MELK expression to control asymmetric cell division. Genetics;
206(4), PubMedID: 28652378
Bornikoel J, Staiger J, Madlung J, Forchhammer K, Maldener I.
(2018)
LytM factor Alr3353 affects filament morphology and cell-cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120 Molecular Microbiology;
PubMedID: 29437253
Quality & Safety
Quality Assurance Statement
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
