TITLE: Plasmid DNA extraction
AUTHORS: Shuning  Guo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to extract plasmid DNA from E. coli.</div></div>

[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.
Centrifuge: 10000 34, 1 min
?. 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.
RNase A must be added to Solution I before use.
?. 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.
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.
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.
Centrifuge: 15000 34, 10 min
?. 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.
Centrifuge: 15000 34, 1 min
?. Discard the filtrate and reuse the collection tube.
?. Add 500 µL HBC Buffer
HBC Buffer must be diluted with isopropanol before use. Please see Page 6 for  instructions.
?. Centrifuge at maximum speed for 1 minute.
Centrifuge: 15000 34, 1 min
?. Discard the filtrate and reuse collection tube.
?. Add 700 µL DNA Wash Buffer.
?. Centrifuge at maximum speed for 1 minute.
Centrifuge: 15000 34, 1 min
?. Discard the filtrate and reuse the collection tube.
?. 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.
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.
Centrifuge: 15000 34, 1 min
This represents approximately 70% of bound DNA. An optional second elution  will yield any residual DNA, though at a lower concentration.
?. Test the concentration and purity of DNA using NanoDrop.
?. Store DNA at -20°C.
?. Repeat step 16~18 once.
?. Suck out the solution from the tube and re-add it to the center of the  column membrane to give a second centrifuge.
Centrifuge: 15000 34, 1 min