实验试剂

Regents Supplied By User

1. Sterile deionized water (or TE buffer)

2. Absolute (96%-100%) ethanol

实验设备

Equipments Supplied By User

1. Centrifuge Capable of 15,000 x g

2. Tubes or vessel capable of 15,000 x g

3. 500mL centrifuge tube.

4. waterbath or heat block preset to 70℃

5. Pipettor

实验步骤

1. Isolate a single colony from freshly streaked selective antibiotic plate and inoculate a starter culture of 2-5mL LB medium containing proper antibiotic. Incubate st 37℃ for about 8 hours with vigorously shaking. Dilute 1.5-3.0 mL starter culture into a 1000-1500 mL selective LB/antibiotic(s) medium and grow at 37°C with agitation for 12-16 h. The culture density should reach 3-4 x 109 per mL. It is strongly recommended that an endA negative strain of E.coli be used for routine plasmid isolation. Examples of such strains include DH5α® and JM109® .

2. Harvest the bacterial cells by centrifugation at 6,000 x g for 15 minutes at 4℃.

Note: If you want to stop the protocol and continue later, discard the medium and freeze the cell pellet at -20℃.

3. Discard supernatant into a waste container. Dry the pellet by placing centrifuge tube upside-down on a paper towel to remove excess media. Add 80 mL of Solution I/RNase A to the bacterial pellet. Resuspend cells completely by shaking or pipetting. Complete resuspension of cell pellet is vital for obtaining good plasmid yields.

4. Add 80 mL Solution II and mix by gently shaking and rotating the tube for 1 minute to obtain a cleared lysate. A 2-3 minutes incubation at room temperature may be necessary. Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.)

Note: do not incubate the lysate over 5 minutes since it can cause permanently denature plasmid.

5. Add 80 mL of Chilled (4℃) Neutralization Buffer and mix by gently shaking and rotating for 1 minute until a flocculent white precipitate forms.

6. Centrifuge at 15,000 x g for 30 minutes at 4℃. Remove the supernatant contains plasmid DNA promptly to a new tube.

7. Carefully transfer the supernatant and clear it again by filtering through filter paper (Whatman #1 or autoclaved coffee filter) into a vessel or tube.

8. Add 500 ul of of Mag-Binds® Particles Solution into each tube and follow by addition of equal volume of MGC Binding Buffer. Mix well by inverting the tube few times.

9. Incubate for 10 minutes at room temperature, mixing few times by inverting the bottle.

10. Magnetize the magnetic particles with a magnet until liquid is cleared.

11. Add 70 mL of SPM Wash Buffe diluted with absolute ethanolr. Invert the tube few times to mix throughly.

12. Magnetize the magnetic particles with a magnet until liquid is cleared.

13. Discard the cleared supernatant. Wash the beads again by repeating step 11-12.

Aspirate the cleared supernatant and air dry the magnetic pellet at room temperature for 5-10 minutes.

14. Elute DNA: Resuspend the Mag-Binds® particles pellet with 2-5 mL Elution Buffer or TE buffer. Incubate at 60℃ for 10 minutes.

15. Centrifuge at 6000 x g for 10 minutes to pellet the Mag-Binds® particles. Transfer the eluted DNA to a clean tube.