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Sterile deionized water
1. Centrifuge with swinging-bucket rotor at room temperature capable of 2000 x g
2. Adapter for 96-well collection plate
3. Vacuum pump or vacuum aspirator capable of achieving a vacuum of 20-24 inches Hg
4. Standard vacuum manifold ( i.e: Omega Product #VAC-03)
5. Vacuum oven or incubator preset to 70°C
1. Culture Volume: Innoculate 1.0-1.3 mL LB/antibiotic(s) medium placed in a 96-well 2mL culture block with E.coli carrying desired plasmid and grow at 37°C with agitation for 20-24 h. 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. Seal the plate with tape or film and pellet bacteria by centrifugation at 1,500 x g for 5 minutes in a swinging-bucket rotor at room temperature.
3. Remove the tape and discard supernatant into a waste container. Dry the plate by tapping the inverted block firmly a paper towel to remove excess media. Add 300ul Solution I/RNase A to the bacterial pellet in each well of the plate. Resuspend cells completely by vortexing and/or pipetting.
No cell clumps should be visible after resuspension of the pellet. Complete resuspension of cell pellet is vital for obtaining good plasmid yields.
4. Add 300ul Solution II into each well. Seal the 96-well Plate with a sheet of sealing film. Mix by gently shaking and rotating the plate for 1 minute to obtain a cleared lysate. A 4 min incubation at room temperature may be necessary. The solution should become viscous and slightly clear. Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.)
5. Remove the sealing film and add 300ul of chilled (4°C ) Neutralization Buffer into each well. Dry the top of the plate with a paper tower. Seal the plate with sealing film and mix by gently inverting the plate for 10 times until a flocculent white precipitate forms.
6. Optional: Place the plate containing the cell lysate in a 92°C water bath for 8 minutes. This heating step denatures and precipitate the proteins and carbohydrates that are not removed by alkaline lysis. This heating step is essential for EndA strains that normally have high level of endonuclease.
7. Optional: Place the plate on ice and incubate for 10 minutes.
8. Assemble the vacuum manifold:
1) Place E-Z 96® Lysate Clearance Plate in the top plate of manifold;
2) Place the 96-well 2 ml plate into the plate holder.
3) Place the top plate of manifold over the base, the 96-well 2 ml Plate now should be positioned under the E-Z 96® Lysate Clearance Plate. (Some manifolds might require internal height adjustment by using an extra small plate.) Seal the unused wells of E-Z 96® Lysate Clearance Plate with film tape.
9. Immediately transfer the lysate into the wells of E-Z 96® Lysate Clearance Plate. Allow the cell lysate to stand for 5 minutes. The white precipitate should float to the top.
10. Apply the vacuum until all the lysate passes through.
11. Turn off the vacuum and discard the E-Z 96® Lysate Clearance Plate. Add 650 ul of isopropanol to the cleared lysates in the 96-well plate, and seal well with sealing film. Immediately mix by inverting several times.
12. Centrifuge the 96-well 2 ml plate at 2,000 x g for 30 minutes at room temperature to precipitate the plasmid DNA. Discard the supernatant by inverting the block, and allow the 96-well 2 ml plate to remain inverted on a paper towel for 1-2 minutes.
13. Desalt the plasmid DNA by adding 600 ul of ice-cold 70% ethanol to each well, and seal each well with sealing film. Centrifuge at 2000 x g for 5 minutes. Discard the supernatant and invert the 96-well 2 ml plate on absorbent toweling to drain off residual solution. Dry the plasmid DNA for 15-20 minutes at room temperature or dry under vacuum for 10 minutes.
14. Add 40-80 ul of 1mM Tris-HCl, pH 8.5 buffer to each well, and seal each well with sealing film. Vortex to resuspend the plasmid DNA.
