Plasmid DNA extraction
Shuning Guo
Abstract
This protocol is used to extract plasmid DNA from E. coli.
Before start
Prepare DNA Wash Buffer, HBC Buffer, and Solution I .
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Add the vial of RNase A to the bottle of Solution I and store at 2-8˚C;
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Dilute DNA Wash Buffer with 100% ethanol 120ml and store at room temperature;
3.Dilute HBC Buffer with isopropanol and store at room temperature;
4.Check Solution II and Solution III for precipitation before use. Redissolve any precipitation by warming to 37˚C.
Steps
Solate 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 hours 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. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
Centrifuge at 10,000 x g for 1 minute at room temperature.
10000x g
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.
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.
Add 350 µL Solution III. Immediately invert several times until a flocculent white
precipitate forms.
Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet
will form. Promptly proceed to the next step.
15000x g
Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
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.
Centrifuge at maximum speed for 1 minute.
15000x g
Discard the filtrate and reuse the collection tube.
Add 500 µL HBC Buffer
Centrifuge at maximum speed for 1 minute.
15000x g
Discard the filtrate and reuse collection tube.
Add 700 µL DNA Wash Buffer.
Centrifuge at maximum speed for 1 minute.
15000x g
Discard the filtrate and reuse the collection tube.
Repeat step 16~18 once.
Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to
dry the column matrix.
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.
Let sit at room temperature for 1 minute.
Centrifuge at maximum speed for 1 minute.
15000x g
Suck out the solution from the tube and re-add it to the center of the
column membrane to give a second centrifuge.15000x g
Test the concentration and purity of DNA using NanoDrop.
Store DNA at -20°C.
