TITLE: Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010)
AUTHORS: New England Biolabs

[DESCRIPTION]
This is the quick version of the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). For the full protocol, please click here.

[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.





[GUIDELINES]
For detailed protocol and more information, visit www.neb.com/T1010

The full protocol is available here.

The video protocol is available here.

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.
 
        PLASMID  REPLICON  COPY  CLASSIFICATION        NUMBER      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    
 
Antibiotics for Plasmid Selection 
ANTIBIOTICCONCENTRATION OFSTOCK SOLUTIONSTORAGETEMP.WORKING CONCENTRATIONAmpicillin100 mg/ml (H2O) –20°C50–200 μg/mlCarbenicillin100 mg/ml (H2O)–20°C20–200 μg/mlChloramphenicol34 mg/ml (ethanol)–20°C25–170 μg/mlKanamycin10 mg/ml (H2O) –20°C10–50 μg/mlStreptomycin10 mg/ml (H2O)–20°C10–50 μg/mlTetracycline5 mg/ml (ethanol)–20°C10–50 μg/ml
 


[STEPS]
1. Pellet 1 mL–5 mL (not to exceed 15 OD units) bacterial culture by centrifugation at 16000 x g for 30 s. Discard supernatant.
 
2. Resuspend pellet in 200 µL.
 

3. Add 200 µL, gently invert tube 5–6 times, and incubate at 4 Room temperature for 1 min. Do not vortex.
 
4. Add 400 µL, gently invert tube until neutralized, and incubate at 4 Room temperature for 1 min. Do not vortex.
 
5. Centrifuge lysate at 16000 x g for 2-5 minutes. 
 

6. Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through.
7. Re-insert column in the collection tube and add 200 µL. Centrifuge for 1 min. 
 
8. Add 400 µL and centrifuge for 1 min.
9. Transfer column to a clean 1.5 ml microfuge tube. 
 
10. Add ≥ 30 µL to the center of the matrix. Wait for 1 min, then spin at 16000 x g for 1 min to elute DNA.