BACs, PACs, P1s and Plasmid Isolation Protocol (standard protocol)
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实验试剂
1. Sterile Deionized Water (or TE Buffer)
实验设备
1. Microcentrifuge capable of at least 18,000 x g (a centrifugal force of 20,000 x g corresponds to 12,000 rpm in a Beckman JA-17 rotor, or 13,000 rpm in a Sorvall SS-34 rotor).
2. Sterile 1.5 ml Centrifuge Tubes
实验步骤
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a starter culture of 2-5 ml LB or YT medium containing the appropriate selective antibiotic. Incubate for ~ 20-24 hr at 37°C with vigorous shaking (~ 300 rpm). Use a flask with a volume at least 4 times the volume of the culture.
2. Pellet 1.5-5 ml bacteria by centrifugation at 13,000 x g for 3 min at room temperature. Decant or aspirate medium and discard.
3. Resuspend the bacterial pellet by adding 200 ul of Buffer T1/RNase A solution, and vortexing. Complete resuspension of cell pellet is vital for obtaining good yields.
4. Add 200 ul of Buffer T2 and gently mix by inverting 5-10 times to obtain a clear lysate. Incubate at room temperature for 5 minutes. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. The lysate should appear viscous. Do not allow the lysis reaction to proceed more than 5 min. (Store Buffer T2 tightly capped when not in use).
5. Add 200 ul of Chilled Buffer T3 and gently mix by inverting 15-20 times until a flocculent white precipitate forms. Incubate on ice for 5 minutes. Do not vortex, as this will result in shearing.
6. Centrifuge at $ 13,000 x g for 10 minutes at 4/C. Promptly proceed to the next step.
7. Carefully transfer the cleared supernatant to a new 1.5 ml tube (not supplied). Add 200ul of BAC Binding Buffer diluted with isopropanol. Invert 3-5 times to mix throughly.
Note: BAC Binding Buffer has to be diluted with isopropanol (96-100%) before use. See the label on the bottle or page 3 for detail instructions.
8. Apply the sample to the HiBind® DNA MicroElute column assembled in a 2 ml collection tube (provided).
9. Centrifuge at > 8,000 x g for 30 second at room temperature. Remove the column, discard the flow-through and re-use the collection tube for next step.
10. Place the column back into the collection tube and add 750ul of SPM Buffer (diluted with ethanol). Centrifuge at > 8,000 x g for 30 seconds at room temperature. Discard the flow-through and re-use the collection tube.
11. Place the column back into the collection tube and centrifuge at maximum speed for 2 minutes to dry the column.
12. Place the HiBind® DNA MicroElute column into a clean 1.5 ml centrifuge tube, apply 20-50 ul of Elution Buffer (10mM Tris-HCl, pH 8.5) or water onto the center of the membrane. Incubate 5 minutes at room temperature.
13. Centrifuge at maximum speed for 2 minutes to elute the DNA.
14. Store the eluted DNA at -20°C.
