Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010) V.4
- New England Biolabs1
- 1New England Biolabs
External link: https://www.neb.com/protocols/2015/12/08/quick-protocol-for-monarch-plasmid-miniprep-kit-t1010
Protocol Citation: New England Biolabs . Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010). protocols.io https://dx.doi.org/10.17504/protocols.io.bg9qjz5wVersion created by Isabel Gautreau
License: This is an open access protocol 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: June 07, 2020
Last Modified: February 25, 2022
Protocol Integer ID: 37904
Keywords: Monarch Kit, DNA, Plasmid, Lysis
Abstract
This is the quick version of the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). For the full protocol, please click here.
Guidelines
For detailed protocol and more information, visit www.neb.com/T1010
General Guidelines:
Yield and quality of plasmid DNA is affected by plasmid copy number, plasmid size, insert toxicity, host strain, antibiotic selection, growth media and culture conditions. For standard cloning strains of E. coli, we recommend using a single colony from a freshly streaked selective plate to inoculate a standard growth media, such as LB (Luria-Bertaini) media. Cultures are typically grown at 37°C and 200–250 RPM in vessels that allow some aeration (Erlenmeyer flasks or culture tubes on a roller drum) and harvested after 12–16 hours as the culture transitions from logarithmic growth to stationary phase. This is the time at which the plasmid DNA content is highest. While cultures in LB often saturate with a final OD600between 3–6, growth to saturation often leads to cell lysis. As a result, plasmid yields and quality are reduced and the likelihood of co-purifying unwanted host chromosomal DNA increases. Use of rich media, such as 2X YT or TB, produces higher biomass in a shorter time period. If chosen for growth, adjustments to the culture times and amount of cells used in the prep should be made to correct for these differences, and to avoid overloading the matrix and reducing DNA yield and quality.
| A | B | C | D | |
| PLASMID | REPLICON | COPY NUMBER | CLASSIFICATION | |
| pUC and its derivatives | pMB1* | > 75 | High copy | |
| pBR322 and its derivatives | pMB1 | 15–20 | Low copy | |
| pACYC and its derivatives | p15A | 10–12 | Low copy | |
| pSC101 | pSC101 | ~5 |
*pUC and its derivatives lack theRopgene and contain a point mutation in the RNAII transcript. These changes result in higher copy number during routine growth with many sources reporting levels as high as 500 copies per cell.
Antibiotics for Plasmid Selection
| ANTIBIOTIC | CONCENTRATION OFSTOCK SOLUTION | STORAGETEMP. | WORKING CONCENTRATION | |
| Ampicillin | 100 mg/ml (H2O) | –20°C | 50–200 μg/ml | |
| Carbenicillin | 100 mg/ml (H2O) | –20°C | 20–200 μg/ml | |
| Chloramphenicol | 34 mg/ml (ethanol) | –20°C | 25–170 μg/ml | |
| Kanamycin | 10 mg/ml (H2O) | –20°C | 10–50 μg/ml | |
| Streptomycin | 10 mg/ml (H2O) | –20°C | 10–50 μg/ml | |
| Tetracycline | 5 mg/ml (ethanol) | –20°C | 10–50 μg/ml |
Materials
MATERIALS
Monarch® Plasmid Miniprep Kit New England BiolabsCatalog #T1010
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
- All centrifugation steps should be carried out at 16000 x g (~13.000 rpm ).
- Add ethanol to Monarch Plasmid Wash Buffer 2 prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2).
- For 50-prep kit add 24 mL of ethanol to 6 mL of Monarch Plasmid Wash Buffer 2
- For 250-prep kit add 120 mL of ethanol to 30 mL of Monarch Plasmid Wash Buffer 2
- If precipitate has formed in Lysis Buffer (B2), incubate at 30 °C –37 °C , inverting periodically to dissolve.
- Store Plasmid Neutralization Buffer (B3) at 4 °C after opening, as it contains RNase A.
Pellet 1 mL –5 mL (not to exceed 15 OD units) bacterial culture by centrifugation at 16000 x g for 00:00:30 . Discard supernatant.
Note
1.5 mL of culture is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).
Resuspend pellet in 200 µL Plasmid Resuspension Buffer (B1) (pink) .
Note
Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
Add 200 µL Plasmid Lysis Buffer (B2) (green) , gently invert tube 5–6 times, and incubate at Room temperature for 00:01:00 . Do not vortex.
Note
Care should be taken not to handle the sample roughly and risk shearing chromosomal DNA, which will co-purify as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.
Add 400 µL Plasmid Neutralization Buffer (B3) (yellow) , gently invert tube until neutralized, and incubate at Room temperature for 00:01:00 . Do not vortex.
Note
Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.
Centrifuge lysate at 16000 x g for 2-5 minutes.
Note
Spin time should not be less than 2 minutes. Careful handling of the tube will ensure no debris is transferred and the 2 minute recommended spin can be successfully employed to save valuable time. For culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.
Carefully transfer supernatant to the spin column and centrifuge for 00:01:00 . Discard flow-through.
Re-insert column in the collection tube and add 200 µL Plasmid Wash Buffer 1 . Centrifuge for 00:01:00 .
Note
Discarding the flow-through is optional. The collection tube is designed to hold 800 μl of flow-through fluid and still allow the tip of the column to be safely above the top of the liquid. Empty the tube whenever necessary to ensure the column tip and flow-through do not make contact.
Add 400 µL Plasmid Wash Buffer 2 and centrifuge for 00:01:00 .
Transfer column to a clean 1.5 ml microfuge tube.
Note
Use care to ensure that the tip of the column does not come into contact with the flow-through. If there is any doubt, re-spin the column for 00:01:00 .
Add ≥ 30 µL DNA Elution Buffer to the center of the matrix. Wait for 00:01:00 , then spin at 16000 x g for 00:01:00 to elute DNA.
Note
Nuclease-free water (7 –8.5 ) can also be used to elute the DNA. Delivery of the Monarch DNA Elution Buffer should be made directly to the center of the column to ensure the matrix is completely covered for maximal efficiency of elution. Additionally, yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated as a result of dilution. For larger plasmids (≥ 10 kb), 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.