E.Z.N.A.?PlasmidMaxiKitSpinProtocol

實驗概要

This Protocol is designed to isolate 500-1200 ug of high Copy-Number plasmids or 50-400 ug of low Copy-Number Plasmids from 200 ml overnight cultures.

實驗步驟

Growth of bacterial culture:

1. Culture volume: Inoculate 200 ml LB/ampicillin (50 ug/ml) medium placed in a 1-4 liter 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 (~300 rpm).Using a flask or vessel with a volume of at 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. We recommend a bacterial density of between 2.0 and 3.0 at OD600. When using untrient-rich media, care should be taken ensure that the cell density does not exceed an OD600 of 3.0.

If using a frozen glycerol stock as inoculum, streak it onto an agar plate containing the appropriate 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 100-200 ml bacteria in appropriate vessels by centrifugation at 3,500-5,000 × 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 12 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. Transfer cell suspension to a 50 ml centrifuge tube capable of withstanding 12,000 ×g (screw-cap polycarbonate or Corex^? glass tubes will suffice). Add 12 ml Solution II, cover, and mix gently but throughly by inverting and rotating tube 10-15 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. Prolonged incubation may lead to nicking of plasmid DNA. (Store Solution II tightly capped when not in use.)

5. Add 16 ml Solution III, cover, and gently mix 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) to pellet the cellular debris and genomic DNA.

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. Increasing centrifugation speed is helpful to completely remove the precipitated bacterial cell material. A tightly packed cell debris pellet indicates efficient lysis.

Note: Step 6 to 12 should be performed in swinging-bucket rotor for maximal plasmid DNA yields. And all centrifugation steps must be carried out at room temperature.

Purify Plasmid DNA with HiBind^TM DNA Maxi Column:

6. Prepare the HiBind Maxi Column. Place a HiBind Maxi Column into a 50 ml collection tube, provided. Add 5 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 assemble the column again in the 50 ml collection tube.

7. Carefully aspirate and add 20 ml of the clear supernatant to the HiBind^? DNA Maxicolumn assembled in the 50 ml collection tube, making sure that no cellular debris is carried over. The Maxicolumn has a maximum capacity of 20 ml. Centrifuge at 3,000-5,000 × g for 3-5 min at room temperature to completely pass lysate through column. Discard the flow-through liquid and repeat this step until the entire sample has been passed through the column. Finally discard the flow-through and reuse the collection tube in Step 8.

8. Add 10 ml Buffer HB to the Maxi column and centrifuge as above. This step ensures that residual protein contamination is removed and must be included for downstream applications requiring high quality DNA. Discard flow-through liquid and reuse the collection tube in the next step.

9. Wash the column by adding 15 ml of DNA Wash Buffer diluted with ethanol. Centrifuge as above and discard flow-through.

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.

10. Optional Step: Repeat wash step with another 10 ml DNA Wash Buffer. Centrifuge as above and discard fluid.

11. Centrifuge the empty capped column for 10-15 min at maxi speed (no more than 6,000 x g) to dry the column matrix.

DO NOT skip this step - it is critical for removing traces of ethanol that may otherwise interfere with downstream applications.

Elution Plasmid DNA From HiBindTM DNA Maxi column:

Optional: For maximal yield and high concentration of plasmid, see alternative protocol of elution on page 7. For fast elution, proceed step 12-13.

12. 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 13.

   2) Bake the column in a vacuum oven or incubator at 65NC for 10 minutes. Remove the column and proceed to Step 13.

13. Place column into a clean 50 ml centrifuge tube. Add 2-3 ml (depending on desired concentration of final product) Elution Buffer (or TE buffer) directly onto the column matrix. Allow column to sit 2 min at room temperature. Centrifuge at max speed (no more than 8,000 x g) for 2 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 is 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 protocol of Elution Plasmid from Column:

1. Place HiBind^TM DNA Maxi column into a clean 50 ml centrifuge tube. Add 6 ml Elution Buffer (or TE buffer) directly onto the column matrix. Allow column to sit 2 min at room temperature. Centrifuge at maxi speed (no more than 6,000 x g) for 5 min to elute DNA.

2. Carefully transfer the eluted plasmid from 50 ml centrifuge tube to a clean tube suitable for precipitation and add 260 ul 5M NaCl and 4.4 ml room temperature isopropanol. Vortex to mix and centrifuge at >15,000 × g for 30 min at 4°C. Carefully decant the supernatant.

3. Wash DNA pellet once with 2 ml ice-cold 70% ethanol and centrifuge at > 15,000 × g for 10 min. Carefully decant the supernatant without disturbing the pellet and air-dry the pellet for 5-10 min.

4. Finally resuspend DNA pellet in 200-500 ul (depending on desired concentration of final product) Elution Buffer or water.