Plasmid Extraction
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:
Growth of the bacterial culture
Harvesting and lysis of the bacteria
Purification of plasmid DNA
We use the OMEGA Bio-tek E.Z.N.A. Plasmid DNA Mini Kit I (D6943-02) for plasmid preparations. Below, we've described the common protocol that is used in our lab. Refer to the manual for other variations on the protocol and more tips.
Preparing Reagents
(First time only)
Add the vial of RNase A to the bottle of Solution I and store at 2-8˚C. (50 and 200 prep size only). We store Solution 1 in the deli fridge (4˚C).
Dilute DNA Wash Buffer with 100% ethanol as described on bottle and store at room temperature. The kit we use calls for 100 mL of 100% ethanol.
Check Solution II and Solution III for precipitation before use. Redissolve any precipitation by warming to 37˚C.
Storage and Stability
All of the E.Z.N.A. Plasmid DNA Mini Kit I and Plasmid DNA Mini Kit II components are guaranteed for at least 12 months from the date of purchase when stored as follows. Solution I (once RNase A is added) should be stored at 2-8˚C. All other materials should be stored at room temperature. Solution II must be tightly capped when not in use.
Vacuum Protocol
All centrifugation should be performed at room temperature unless otherwise noted. For low copy number plasmids refer to Page 21. This protocol is designed to isolate plasmid DNA from E. coli grown in an overnight 1-5 mL LB culture. See Page 7 for guidelines on preparing the vacuum manifold used in this protocol.
Materials and Equipment to be Supplied by User:
Vacuum Manifold
100% ethanol
Microcentrifuge capable of at least 13,000 x g
Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes
Appropriate centrifuge and centrifuge tube for Step 1
Optional: sterile deionized water
Optional: water bath or incubator capable of 70°C
Optional: 3M NaOH solution
Before Starting:
Heat Elution Buffer to 70°C if plasmid DNA is >10 kb.
Prepare DNA Wash Buffer and Solution I according to the instructions above.
Steps:
Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hr at 37°C with vigorous shaking (300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture.
Centrifuge at 10,000 x g for 1 minute at room temperature.
Decant or aspirate and discard the culture media.
Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents above.
Transfer suspension into a new 1.5 mL microcentrifuge tube.
Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.
Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate forms.
Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
Prepare the vacuum manifold according to manufacturer’s instructions.
Connect the HiBind DNA Mini Column to the vacuum manifold.
Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column.
Turn on the vacuum source to draw the sample through the column.
Turn off the vacuum.
Add 500 μL HB Buffer.
Turn on the vacuum source to draw the buffer through the column.
Turn off the vacuum.
Add 700 μL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. See Preparing Reagents section above.
Turn on the vacuum source to draw the buffer through the column.
Turn off the vacuum.
Repeat Steps 17-19 for a second DNA Wash Buffer wash step.
Transfer the HiBind DNA Mini Column to a 2 mL Collection Tube.
Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
Note: It is important to dry the HiBind DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.
Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
Note: We use deionized water (ddH2O).
Note: The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.
Let sit at room temperature for 1 minute.
Centrifuge at maximum speed for 1 minute.
Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
Store DNA at -20°C.
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