Team:XJTLU-CHINA/Protocols Isolation
Isolation of plasmid in E.coli DH5ɑ
Reagents:
AxyPrep Plasmid Miniprep Kit (Miniprep column; 2 ml Microfuge tube; 1.5 ml Microfuge tube; Resuspension Buffer S1 (contains RNase A); Lysis Buffer S2; Neutralization Buffer S3; Wash buffer Buffer W1; Desalting Buffer W2; Eluent Buffer (2.5 mM Tris-HCl))
Procedure:
- Collect 4 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the E. coli. Decant or pipette off as much of the supernatant as possible.
- Re-suspend the bacterial pellet in 250 μl of Buffer S1 by vortex. Please be sure that the bacteria are completely re-suspended before proceeding.
- Add 250 µl of Buffer S2, and mix by gently inverting the tube 4-6 times.
- Add 350 µl of Buffer S3, and mix by gently inverting 6-8 times. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
- Insert the required AxyPrep Plasmid Miniprep column into the fittings on the 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column.
- Add 500 µl of Buffer W1 to Miniprep column. Centrifuge at 12,000×g for 1 minute.
- Pipette 700 µl of Buffer W2 along the wall of the column to remove residual salt. Centrifuge at 12,000×g for 1 minute. Repeat this wash step with a second 700 µl aliquot of Buffer W2.
- Centrifuge at 12,000×g for 1 minute to purge residual Buffer W2 from the binding membrane.
- Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. To elute the purified plasmid DNA, add 60~80 µl of Eluent buffer to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.
Application in this project : pSB1C3-BBa_K515005 extraction from DH5ɑ transformant
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