May 21, 2020
Plasmid DNA Mini Kit I- Spin Protocol
This protocol is a draft, published without a DOI.
- 1BYU
Protocol Citation: Alex Zegarra 2020. Plasmid DNA Mini Kit I- Spin Protocol . protocols.io https://protocols.io/view/plasmid-dna-mini-kit-i-spin-protocol-bgq9jvz6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 21, 2020
Last Modified: May 21, 2020
Protocol Integer ID: 37377
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.
4) Rinse with 200 uL of Neutralization buffer (P3)
(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.