實驗概要
The E.Z.N.A.TM Fastfilter Plasmid Midi Kit combines the power of HiBind? technology with the time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality plasmid DNA. Omega Bio-Tek!s midi columns facilitate the binding, washing, and elution steps, thus enabling multiple samples to be simultaneously processed. This kit also include a special filter cartridge, which replaces the centrifugation step following alkaline lysis. Following lysis the DNA is bound to the silica membrane and contaminants are removed with a simple wash step.
實驗步驟
1. Culture volume: Inoculate 30-50 ml LB/ampicillin (50 ug/ml) medium placed in a 200-400 milliliter culture flask with E.coli carrying desired plasmid and grow at 37°C with agitation for 12-16 h. For best results use overnight culture as the inoculum. 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?.
Optimal growth conditions of bacteria is vital of obtaining maximal plasmid DNA yields. The best conditions are achieved by picking a single isolated colony from a freshly transformed or freshly plate to inoculate a 2-5ml starter culture containing the appropriate antibiotic. Incubate for ~8hr at 37°C with vigorous shaking (~300rpm). Then used to inoculate appropriate volume of Pre-warmed liquid growth medium with antibiotic. Grow at 37°C for 12-16 hr with vigorous shaking(~300rpm).Using a flask or vessel with a volume of a least 3-4 times the volume of the culture and dilute the starter culture 1/500 to 1/1000 into growth medium.
Following overnight bacterial growth, an OD600 of 1.5~2.0 indicates a well-grown culture. For the best result determination of OD600 for each culture is recommended. it is important to dilute the bacterial culture (10 to 20 fold) to enable photometric measurement in the linear range between 0.1 and 0.5 OD600. For maximal yields, the OD600 of cultures should be under 3.0.
If using a frozen glycerol stock as inoculun, streak it onto an agar plate containing the apropriate antibiotic for single colony isolation. Then picking a single colony and inoculate the 2-5ml starter culture as described above.
Lyse bacterial cells with Alkaline-SDS Solution:
2. Pellet up to 30-50 ml bacteria in appropriate vessels by centrifugation at 3,500-5,000 x g for 10 min at room temperature.
3. Decant or aspirate medium and discard. To ensure that all traces of the medium are removed, use a clean paper towel to blot excess liquid from the wall of the vessel. To the bacterial pellet add 2.5 ml Solution I/RNase A. Resuspend cells completely by vortexing or pipetting up and down. Complete resuspension of cell pellet is vital for obtaining good yield.
4. Add 2.5 ml Solution II, cover, and mix gently but throughly by inverting and rotating tube 7-10 times to obtain a cleared lysate. A 5 min incubation at room temperature may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Prolonged incubation may lead to nicking of plasmid DNA. (Store Solution II tightly capped when not in use.)
5. Add 1.25 ml ice-cold Buffer N3, cover, and gently mix by inverting tube several times until a flocculent white precipitate forms. Prepare a Lysate Clearance Filter Syringe by placing the barrel in a tube rack to keep the syringe upright.
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. Add 2 ml Buffer GBT, cover and gently mix by inverting tube 3-4 times.
7. Prepare the HiBind Midi Column. Place a HiBind Midi Column into a 15 ml collection tube, provided. Add 2 ml of Buffer GPS to the column and Lit it sit at room temperature for 3-10 min. Spin in a swinging bucket rotor at 3,000-5,000 x g for 5 minutes at room temperature. Discard the eluate and assembled the column again into the 15 ml collection tube.
Clear the lysate with Lysate Clearance Filter Syringe:
8. Immediately pour the lysate into the barrel of the Lysate Clearance Filter Syringe. Allow the cell lysate to sit for 2 minutes. The white precipitate should float to the top. Use a new 15 ml tube to collect the cell lysate. Insert the plunger back into the barrel of the syringe.
9. Hold the Lysate Clearence filter syringe barrel over the 15 ml tube and gently insert the plunger to expel the cleared lysate to the tube.
Note: Some of the lysate may remain in the flocculent precipitate, do not force this residual lysate through the filter. Alternatively, the cell debris and KDS-precipitation can be removed by centrifugation at $12,000 x g for 10 min at 4°C, instead of using Clearance Filter Syring in step 8-9. A tightly packed cell debris pellet indicates efficient lysis. Using this alternatively cleared step may improve the yield because all of the solution can be collected comparing with Lysate Clearance Filter Syringe.
Note: Step 10 to 16 should be performed in swinging-bucket rotor for maximal plasmid DNA yields. All of centrifugation steps must be carried out at room temperature.
10. Prepare the vacuum manifold according to manufacturer’ s instructions and connect the midi column to the manifold.
11. Transfer the cleared lysate into the HiBind? DNA Midi column, be careful not to overfill the column, apply the vacuum to allow all sample pass through the column. Repeat to transfer the lysate into the column until the entire sample has been passed through.
12. Add 3.0 ml Buffer HB to the column and apply the vacuum to draw the liquid through the column.
13. Wash the column: add 3.5 ml of DNA Wash Buffer (pre-diluted with absolute ethanol) into the column and allow it to pass through the column.
14. Wash the column again with 3.5 ml DNA Wash Buffer by repeating step 5.
15. Keep the vacuum on for another 10 minutes after the liquid pass through the column. (This step to ensure the removal of residue ethanol).
16. Centrifuge the empty capped column for 10-15 min at maxi speed (no more than 8,000 x g) to dry the column matrix. Remove any traces of ethanol from the column’s inner surface or O-ring using a pipette.
17. Further Drying The Column (Optional). Choose either of the methods below to further dry the column before eluting DNA (only if necessary):
1) Place the column into a vacuum container to dry the ethanol for 10 minutes. Then, remove the column and place into a vacuum chamber at room temperature. Any device connected to a vacuum source may be used. Seal the chamber and apply vacuum for 15 min. Remove the column and proceed to Step 16.
2) Bake the column in a vacuum oven or incubator at 65°C for 10 minutes. Remove the column and proceed to Step 16.
18. Place column into a clean 15 ml centrifuge tube. Add 0.5-1.0 ml (depending on desired concentration of final product) Elution Buffer (or water) directly onto the column matrix. Allow column to sit 2 min at room temperature. Centrifuge at maxi speed (no more than 8,000 x g) for 3-5 min to elute DNA. This represents approximately 60-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. Alternatively, a second elution may be performed using the first eluate to maintain a high DNA concentration. Also, preheating the water to 70°C prior to elution may significantly increase yields.
Note: The plasmid DNA obtained using this protocol performs well in PCR, restriction digests, lipid mediated transfection and transformation. The expected concentration of plasmid vary between different copy number vector. However, the concentration of high copy-number plasmid is 150-400ug/ml. Some residual ethanol is present, but does not interfere with these downstream applications. One may get high concentration and absolutely remove ethanol with optional elution step as following.
Alternative of Elution Step from Column:
1. Place HiBindTM DNA Midi column into a clean 15 ml centrifuge tube. Add 3 ml Elution Buffer (Water) directly onto the column matrix. Allow column to sit 2 min at room temperature. Centrifuge for 3-5 min at maxi speed to elute DNA.
2. Carefully transfer eluted plasmid from 15 or 30 ml centrifuge tube to a clean tube suitable for precipitation. add 130 ul 5M NaCl and 2.2 ml room temperature isopropanol. Vortex to mix and centrifuge at >15,000 x g for 30 min at 4°C. Carefully decant the supernatant.
3. Wash DNA pellet once with 1 ml ice-cold 70% ethanol and centrifuge at > 15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet. Air-dry the pellet for 5-10 min.
4. Finally resuspend DNA pellet in 200ul-500ul (depending on desired concentration of final product) Elution Buffer or water.