Oct 03, 2021
Version 3
Plasmid DNA extraction V.3
- 12021 iDEC NEFU_China
- NEFU_China 2021
Protocol Citation: Shuning Guo 2021. Plasmid DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.byqzpvx6Version created by Shuning Guo
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: October 03, 2021
Last Modified: October 03, 2021
Protocol Integer ID: 53753
Keywords: Pasmid
Abstract
This protocol is used to extract plasmid DNA from E. coli.
Guidelines
This protocol is based on E.Z.N.A.® Plasmid DNA Mini Kit I Protocol - Spin Protocol.
Materials
• 100% ethanol
• Isopropanol
• Microcentrifuge capable of at least 13,000 x g
• Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes
• Culture tubes
• Optional: sterile deionized water
• Optional: water bath or incubator capable of 70°C
• Optional: 3M NaOH solution
Safety warnings
Please wear gloves before start.
Before start
Prepare DNA Wash Buffer, HBC Buffer, and Solution I .
1. Add the vial of RNase A to the bottle of Solution I and store at 2-8˚C;
2. 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.
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.
10000 x g, Room temperature, 00:01:00
1m
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.
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.
15000 x g, Room temperature, 00:10:00
10m
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.
15000 x g, Room temperature, 00:01:00
1m
Discard the filtrate and reuse the collection tube.
Add 500 µL HBC Buffer
Note
HBC Buffer must be diluted with isopropanol before use. Please see Page 6 for
instructions.
Centrifuge at maximum speed for 1 minute.
15000 x g, Room temperature, 00:01:00
1m
Discard the filtrate and reuse collection tube.
Add 700 µL DNA Wash Buffer.
Centrifuge at maximum speed for 1 minute.
15000 x g, Room temperature, 00:01:00
1m
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.
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.
15000 x g, Room temperature, 00:01:00
Note
This represents approximately 70% of bound DNA. An optional second elution
will yield any residual DNA, though at a lower concentration.
1m
Suck out the solution from the tube and re-add it to the center of the
column membrane to give a second centrifuge.15000 x g, Room temperature, 00:01:00
1m
Test the concentration and purity of DNA using NanoDrop.
Store DNA at -20°C.