TITLE: Plasmid DNA Mini Kit I- Spin Protocol 
AUTHORS: Alex  Zegarra

[STEPS]
?. 1) 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 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®.
?. 2) Centrifuge at 10,000 x g for 1 minute at room temperature.
?. 3) Decant or aspirate and discard the culture media.
?. 4) 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 section.
?. 5) Transfer suspension into a new 1.5 mL microcentrifuge tube.
?. 6) 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.
?. 7) 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.
?. 8) Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
?. 9) Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.(BREAK)
?. Preparing Column Prior to use (non-optional)Protocol for Column Equilibration:1) Add 100 μL 3M NaOH to the HiBind® DNA Mini Column.
?. 2) Centrifuge at maximum speed for 30-60 seconds.
?. 3) Discard the filtrate and reuse the collection tube.
?. (CONTINUATION of Step 9) 10) 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.
?. 11) Centrifuge at maximum speed for 1 minute.
?. 12) Discard the filtrate and reuse the collection tube.
?. 13) Add 500 μL HBC Buffer.
?. 14) Centrifuge at maximum speed for 1 minute.
?. 15) Discard the filtrate and reuse collection tube.
?. 16) Add 700 μL DNA Wash Buffer.Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use
?. 17) Centrifuge at maximum speed for 1 minute.
?. 18) Discard the filtrate and reuse the collection tube.Optional: Repeat Steps 16-18 for a second DNA Wash Buffer wash step.
?. 19) 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.
?. 20) Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
?. 21) Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane. 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.
?. 22) Let sit at room temperature for 1 minute.
?. 23) 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.
?. 24) Store DNA at -20°C.
?. 4) Rinse with 200 uL of Neutralization buffer (P3)