Miniprep Plasmid DNA Purification from E. Coli
Introduction
Materials
- Equipment
- Microcentrifuge capable of holding 1.5mL tubes and producing at least 17,900 x g
- Sterile 1.5mL microcentrifuge tubes
- Pipettes for pipetting volume ranges from 1 μL to 1000 μL
- Sterile pipette tips
- QIAprep Spin Miniprep Kit:
- QIAprep Spin Columns
- QIAprep Collection tubes
- QIAprep buffers P1, P2,N3, PB, PE (note on location: P1 stored in Chilly, very left on top shelf of fridge door)
- Elution Buffer (QIAprep EB Buffer or alternative)
- If Miniprep Kit is brand new:
- LyseBlue®, RNase A
- 96–100% ethanol
Procedure
A A | |
1 1 | Samples’ names and storage location |
2 2 | Unexpected and expected observations and results (either in writing or in picture form) |
3 3 | Volume of bacterial culture used for lysate |
4 4 | Batch number of your kit |
5 5 | Clarification notes (protocol modifications, issues, significant notes) |
6 6 | Elution buffer used and volume |
7 7 | Measured concentration of samples |
Important Notes
- Verify that RNase A solution (provided in the kit) has been added to Buffer P1 before use.
Buffer P1 that has had RNase A added should be clearly marked as such with a black X on the top, with a date of addition written.
If adding it yourself: Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 µg/ml. Mix and store at 2–8°C
- Verify that LyseBlue reagent (provided in the kit) has been added to Buffer P1 before use.
Buffer P1 that has had LyseBlue reagent added should be clearly marked as such with a black X on the top, with a date of addition written.
If adding it yourself: Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 to achieve the required working concentration (e.g., 10µL LyseBlue into 10mL buffer P1). Make sufficient LyseBlue/Buffer P1 working solution for the number of plasmid preps being performed.
- LyseBlue precipitates after addition into Buffer P1. This precipitate will completely dissolve after addition of Buffer P2. Shake Buffer P1 before use to resuspend LyseBlue particles.
- Verify that ethanol (96–100% purity) has been added to Buffer PE before use (see bottle label for volume)
Buffer PE that has had ethanol added should be clearly marked as such with a black X on the top, with a date of addition written.
If adding it yourself: Use the proper purity of ethanol.
- Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37°C. Do not shake Buffer P2 vigorously.
- Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2 in the air.
- Buffer P1 is stored in the 4°C Fridge after the addition of RNase A. Keep Buffer P1 in the fridge until needed, and store immediately after use.
- Buffers P2, N3 and PB contain irritants. Wear gloves when handling these buffers.
- SoundBio Lab often grows 2mL overnight cultures.
- All centrifugation steps are carried out at 17,900 x g (rcf) in a conventional table-top microcentrifuge at room temperature.
- Always ensure the centrifuge is balanced properly.
- When working with multiple samples, it is important to label all containers used to keep track of centrifuged tubes, including the collection tubes.
- Elution of pure DNA with ≤50µL requires the buffer to be added directly to the center of the membrane.
Procedure
- Harvest bacterial cells by adding 1–5mL overnight culture in sterile labelled 1.5mL microcentrifuge tubes.
The total volume of culture will need to be pelleted in steps if the volume is above 1.5mL.
- Transfer 1.5mL of culture into the 1.5mL microcentrifuge tube.
- Centrifuge for 3 min.j
- Discard supernatant.
- Repeat steps 15 to 17 as necessary until the entire overnight culture is processed.
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and vortex vigorously until no cell clumps remain.
CRITICALNo cell clumps should be visible after resuspension of the pellet.
- Add 250 μl Buffer P2 and mix thoroughly but gently by inverting the tube 4–6 times.
CRITICALDo not vortex, because this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.
CRITICALDo not allow the lysis reaction to proceed for more than 5 min.
If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions, or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
- Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times.
CRITICALTo avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3.
The solution should become cloudy. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
- Centrifuge for 10 min.
A compact white pellet will form in a clear supernatant
PAUSEIf the pellet does not seem firm or is still very cloudy, additional centrifugation time may be required.
CRITICALWhile this centrifugation is in process, perform step 22.
- Meanwhile, place a QIAprep 2.0 spin column in a provided 2mL collection tube.
Make sure all tubes are labelled. It is recommended to label both your spin column and your collection tube.
- To bind the plasmid DNA, apply 800 μl of the supernatant from step 22 to the QIAprep 2.0 spin column by pipetting and centrifuge for 30-60 s.
- Discard the flow-through. Place the QIAprep 2.0 spin column back into the same collection tube.
- Recommended: Wash the QIAprep 2.0 spin column by adding 500 μl Buffer PB and centrifuging for 30–60 s. Discard the flow-through and place the QIAprep 2.0 spin column back into the same collection tube.
endA- host strains such as XL-1 Blue and DH5α® do not require this additional wash step. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
- Wash the QIAprep 2.0 spin column by adding 750 μl Buffer PE and centrifuging for 30–60 s.
- Discard the flow-through and place the QIAquick column back into the same collection tube.
- Centrifuge the QIAprep 2.0 spin column once more for 1 min to remove residual wash buffer.
Residual wash buffer will not be completely removed unless the flowthrough is discarded before this additional centrifugation. This step ensures any residual ethanol, which may interfere with subsequent enzymatic reactions, is removed.
- Properly label a new sterile 1.5mL microcentrifuge tube with sample details, your initials and date. Place the QIAquick column from the previous step into the labelled tube.
- To elute DNA, add 30 μl Buffer EB (10mM Tris·Cl, pH 8.5) to the surface of each QIAprep 2.0 spin membrane, let the column stand for 1 min and centrifuge for 1 min.
It's ok if the column stands for more than 1 minute. Just don't let it sit there long enough for it to dry out.
Storage + Cleaning
- If the purified plasmid DNA is NOT to be immediately used, temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box. Properly note where it was stored.
This protocol describes the method used for silica-membrane-based column purification of up to 20 μg of high-copy plasmid DNA from 1–5mL overnight cultures of E. coli in LB medium using the QIAprep Spin Miniprep Kit-Qiagen. For purification of low-copy plasmids and cosmids, large plasmids (>10kb), and DNA prepared using other methods, refer to SOP No. SBL xxx.
QIAprep Miniprep procedure is based on a series of sequential steps involving the release of plasmid DNA from bacterial cells followed by the purification of the plasmid DNA, as follows:
Release of plasmid DNA
1) Resuspension of harvested bacterial cells in buffer P1
2) Alkaline lysis of bacterial cells with buffer P2 in the presence of SDS and RNase A with the consequent release of plasmid DNA and denaturation of chromosomal DNA and cell proteins
3) Neutralization of the bacterial lysate and adjustment to high-salt–binding conditions with buffer N3 with the consequent precipitation of denatured proteins, chromosomal DNA, cellular debris and SDS
4) Clearing of bacterial lysate by centrifugation
Purification of plasmid DNA
1) Selective adsorption of plasmid DNA onto the silica membrane in the QIAprep spin column under high-salt conditions provided by the binding buffer PB
2) Removal of endonucleases with buffer PB
3) Removal of salts with the ethanol-containing Buffer PE
4) Elution of high-quality plasmid DNA under low-salt and basic pH conditions with a small volume of elution buffer EB.