E.Z.N.A.® Endo-free Plasmid Mini Kit I Spin Protocol
互联网
实验步骤
1. Inoculate 5ml LB/ampicillin (50 ug/ml) medium placed in a 10-20 ml culture tube with E.coli carrying desired plasmid and grow at 37°C with agitation for 12-16 h. It is strongly recommended that an endA negative strain of E.coli be used for routine plasmid isolation. Examples of such strains include DH5"® and JM109®.
2. Pellet 1.5-5 ml bacteria in appropriate vessels by centrifugation at 10,000 × g for 1 min at room temperature.
3. Decant or aspirate medium and discard. To the bacterial pellet add 250 u Solution I/RNase A. Resuspend cells completely by vortexing or pipetting up and down. Complete resuspension of cell pellet is vital for obtaining good yields.
4. Add 250 u Solution II and mix gently but throughly by inverting and rotating tube 4-6 times to obtain a cleared lysate. A 2 min incubation at room temperature may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.)
5. Add 125 u ice-cold Buffer N3 and mix gently but throughly by inverting tube several times until a flocculent white precipitate forms. Centrifuge at $12,000 × g for 10 minutes at room temperature (preferably at 4°C).
Note: The Buffers must be mixed throughly. If the mixture appears still viscous, brownish and conglobated, more mixing is required to completely neutralize the solution. Complete neutralization of the solution is vital of obtaining good yields.
6. CAREFULLY aspirate and transfer the cleared supernatant to a clean 1.5ml centrifuge tube. Add 0.1 volume of ETR Solution to the cleared lysate. Mix by inverting tube 7-10 times and incubate on ice for 10 minutes. Invert the tube serval times during the incubation.
Note: After addition of ETR Solution, the lysate should appear turbid, but it should become clear after incubation on ice.
7. Incubate the lysate at 42°C for 5 minutes. The lysate should appear turbid again. Centrifuge at 12,000 × g for 3 minutes at 25°C. The ETR Solution will form a blue layer at bottom of tube.
8. Transfer the top aqueous phase (cleared lysate) into a new 1.5 ml tube and add 0.5 volume of absolute ethanol (room temperature, 96-100%) and gently mix by inverting tube 6-7 times. Incubate at room temperature for 1-2 minutes.
9. Transfer 700 u of the mixture(from Step 8) into a clean HiBindTM DNA Mini Column assembled in a 2 ml collection tube(provided). Centrifuge at 10,000 × g for 1 min at room temperature to pass solution through column.
10. Discard the flow-through and load the remaining of the mixture into the column and centrifuge as above. Discard the flow-through and re-use the collection tube.
11. Wash column with 500 u Buffer HB and centrifuge as above. This step ensures that residual protein contamination is removed and must be included for downstream application requiring high quality DNA.
12. Discard the flow-through liquid and wash the column by adding 700 u DNA Wash Buffer diluted with ethanol. Centrifuge as above and discard flowthrough.
Note: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated. DNA Wash Buffer must be brought to room temperature before use.
13. Repeat wash step 12 with another 700 u DNA Wash Buffer diluted with ethanol.
14. Discared the flow-through liquid. Centrifuge the empty column at maximum speed ($13,000×g) for 2 min to dry the column matrix. Do not skip this step-it is critical for removing ethanol from the column.
15. Place column into a new clean 1.5 ml micro-centrifuge tube. Add 30-50 u (depending on desired concentration of final product) Endotoxin-Free Elution Buffer (or water) directly onto the column matrix and centrifuge at $13,000 × g for 1 min to elute DNA. This represents approximately 70-85% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
