Plasmid DNA Maxiprep Protocol
Tengyun Ma Edited in 3/29/2021
1.Kit
| M5 HiPer Multi-color EndoFree Plasmid Maxi Kit | 10T | MF032-01 |
|---|---|---|
2.Before Starting
Bacterial cultures
Grow transformed E. coli cells overnight in LB (Luria-Bertani) medium with the appropriate antibiotic. Harvest the bacterial culture in transition between exponential phase and stationary phase. The culture should have a cell density of ~109 cells/mL or an optical density of 2.0 at 600 nm (OD600). (12-16h)
Plasmid type and copy number
Recommended volumes of cell culture for plasmid DNA purification are listed in the table below.
| Plasmid Copy Number | Miniprep | Midiprep | Maxiprep | |
|---|---|---|---|---|
| High-copy plasmid | 1–3 mL | 15–25 mL | 100–200 mL | |
| Low-copy plasmid | 10–15 mL | 25–100 mL | 250–500 mL |
Verify that RNase A is added to the Solution1.
Warm Solution 2 and Solution 3 briefly at 37°C to redissolve any particulate matter. Do not shake bottle.
3.Materials needed
- Overnight culture of transformed E. coli cells
- Isopropanol
- Ethanol absolute
- Sterile, microcentrifuge tubes
- Tubes or centrifuge bottles for harvesting cells
- Centrifuge and rotor appropriate for harvesting cells
- Sterile 50mL centrifuge tube (elution tube) capable of withstanding centrifugation forces >12,000 × g
- Centrifuge capable of centrifuging at >12,000 × g at 4°C
- Timer
- Pipettor (1000ul ) and tips
4.Components supplied with the kit
5.Steps
| 1 | Equilibrate | Add 2.5 mL Buffer BL to the MaxiSpin Column with collection tube,10000 rpm for 2 min. Remove all medium. |
|---|---|---|
| 2 | Harvest | Sediment E. coli cells by centrifugation at10000 rpm for 2 min. Discard all medium. |
| 3 | Resuspend | Add 10 mL Solution1 with RNase A to the cell pellet and resuspend the pellet until it is homogeneous reddish-brown. |
| 4 | Lyse | Add 10 mL Solution2. Mix gently by inverting the capped tube until the mixture is homogeneous clear purple-red. Do not vortex. Incubate at room temperature for 5 minutes. Note: Do not allow lysis to proceed for more than 5 minutes. |
| 5 | Precipitate | Add 10 mL Solution3. Mix immediately by inverting the tube until the mixture is homogeneous yellow. Do not vortex. Incubate at room temperature for 2 minutes. Centrifuge the lysate at 10000 rpm for 10 minutes at room temperature. Add 0.3x Isopropanol，mix up and down. Note: If the pellet does not adhere to the bottom of the tube, incubate the tube at room temperature for 5 minutes to allow the lysate and gelatinous pellet to separate. Pipet the clear lysate into another tube and centrifuge the tube at 10000 rpm for 5 minutes at room temperature to remove any remaining cellular debris. |
| 6 | Bind | Load the supernatant onto the equilibrated column. Allow the solution in the column standing for 2 min. Centrifuge the tube at 10000 rpm for 1 min. Remove all medium.(Less than 10ml per time) |
| 7 | Wash | Add 5 mL ToxinOut Buffer to the column. Standing for 10 min. Centrifuge the tube at 10000 rpm for 1 min. Discard the flow‑through after ToxinOut Buffer drains from the column. Add 10 mL Wash Buffer (WB2) to the column. Centrifuge the tube at 10000 rpm for 1 min. Discard the flow‑through after Wash Buffer (WB2) drains from the column. Centrifuge the tube at 10000 rpm for 5min to shake off the residual liquid. |
| 8 | Elute | Place a new sterile 50-mL centrifuge tube under the column. Leave it for 5 minutes and let the ethanol evaporate completely. Add 1~2 mL (500ul) Elution Buffer (Buffer EB) to the column. Standing for 2 min. Centrifuge the tube at 10000 rpm for 1 min. The elution tube contains the purified DNA. |
| 9 | Wash | In order to increase the elution efficiency, Repeat step 8 once. |
| 10 | Determination | Nano Drop detects DNA concentration. To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at –20°C for long-term storage. |
备注：OD260值为1相当于大约50 μg/ml双链DNA。纯化的质粒DNAOD260/OD280通常在1.8-2.0左右，可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的实验中。