Growing cells:

1. Grow a 5 ml culture of cells in the presence of antibiotic - start this from a single colony picked from a plate.
2. After approximately 6 to 8 hours (solution should be quite cloudy) dump the 5 ml culture into 1 liter of LB broth containing antibiotic - grow overnight with shaking at 37^oC.
To insure good aeration, use a 4 liter flask for 1 liter of broth.

Preparation of plasmid from 1 liter of cells (scale all volumes proportionately if you are starting with a smaller volume of cells):

Solutions for cell lysis:

• 30 ml Solution 1: 50 mM Glucose/25 mM Tris-Cl pH 8.0/10 mM Na₂EDTA. Leave at ~20^oC.
• 60 ml Solution 2: 0.2 M NaOH/1% SDS (prepare this fresh). Leave at ~20^oC.
• 45 ml Solution 3^§: 26.6 ml of 5M potassium acetate + 5.3 ml of glacial acetic acid + 13.1 ml of water. Chill on ice.
Solution 3 has often been a source of error in this protocol. The 5 M potassium acetate is combined with glacial acetic acid so the final pH is acidic. This is essential so that solution 3 neutralizes the NaOH from solution 2. Mistakes occur when people do not realize whether the potassium acetate solution they are using has already had the pH adjusted. Also note that there is often a brown precipitate that forms in the 5 M potassium acetate. This doesn't interfere with the plasmid preparation.

Procedure: (*signifies points where the preparation can be left overnight.)

1. Collect cells at 8000 rpm for 10' in SLA 1500 rotor. 200 ml portions of culture can be added and centrifuged sequentially in the same bottle so that the cells are collected in one or two bottles. Only fill the bottles to within 2cm of the top; if they are filled higher, the excess leaks out during centrifugation.
2. Resuspend cells in 30 ml solution 1 at ~20^oC. Triterate the sample with a pipette so the cells are completely dispersed.
3. Add 60 ml solution 2, cap and gently invert several times to mix. Avoid vigorous mixing so the chromosomal DNA is not sheared; shearing may lead to greater contamination from chromosomal DNA. Leave the sample on ice for 5'.
4. Remove cap and add 45 ml of ice cold solution 3. Recap and gently mix by inverting the bottle. Place bottle on ice and gently invert intermittently over a period of at least 30'.
5. Centrifuge at 12000 rpm for 20' in SLA 1500 rotor.
6. Place a wad of rayon securely in the stem of a small funnel and mount the funnel on top of a clean 250 ml bottle. Filter the supernatant from the preceding step through the rayon and collect in the 250 ml bottle.
7. Add 200 ug of RNase A and digest RNA for 1 hour at room temperature.
8. Add 0.6 volumes of isopropanol, mix by inverting several times, and incubate 15' at 20^oC
9. Collect nucleic acid at 10000 rpm for 10' in SLA 1500 rotor.
10. Rinse pellet with 5 to 10 ml of cold 75% Ethanol. If the precipitate remains in place, remove the ethanol with a pipette. If not, spin 10000 rpm for 10' in the SLA 1500 and then remove the ethanol.
11. Dissolve pellet in 10 ml of TE. This can be speeded up by triterating the sample with a pipette.
12. Remove debris at 10000 rpm for 10' in the SLA 1500 rotor. Because of the angle of the bottle in the rotor, the debris should collect on the upper side of the tube and the desired solution should fall to the lower side of the tube. Maintain the angled orientation of the bottle as it is withdrawn from the rotor.

Phenol Extraction.

1. Transfer the supernatant to a polypropylene Sorvall tube that has a purple cap assembly. Add 10 ml of phenol (previously equilibrated according to Maniatis), tightly cap the tube, and shake for about 1'. Centrifuge at room temperature for 10' in the HB4 rotor at 10,000 rpm. Although phenol/chloroform combinations will work, phenol alone is recommended for the first extraction because it removes more impurities.
2. After centrifuging, transfer as much of the aqueous phase (top phase) to a new Sorval tube. Avoid transfering the white material at the aqueous/organic interface. Discard the phenol layer in an appropriate waste bottle usually located in the hood.
3. Extract a second time with 10 ml of phenol/chloroform/isoamyl alcohol (49.5:49.5:1). This mixture leaves less phenol in the aqueous phase than extracting with phenol alone. If too much phenol is left in the aqueous phase when the PEG is added (step 18), the DNA collects as an oil instead of as a precipitate.
4. Extract the aqueous layer with 10 ml of ether, shaking and centrifuging as described above. Remember that ether is highly flammable so use it in the hood as much as possible. Make sure there are no nearby flames.
5. After separating phases by centrifugation (10', 10,000 rpm, HB4), transfer the ether (top phase) to a waste bottle in the hood. Transfer the aqueous layer into a clean 40 ml sorvall centrifuge tube, which can receive a purple cap.
6. Heat the tube of DNA at 37 ^oC for about 15' to evaporate residual ether.

PEG precipitation.

1. Add 2 ml of 50% PEG 8000 and 1 ml of 5 M NaCl. (The PEG stock solution must be made up well ahead of time in order to allow the PEG to dissolve.)  Cap the 40 ml tube and mix thoroughly by inverting the tube.
2. * Leave sample on ice for > 2hr (refrigerate if left overnight). Do not put the sample in the freezer; the sample will freeze and the precipitation is likely to fail. If a large precipitate is apparent after 1 hour, it is OK to proceed with the next step.
3. Spin sample at 15,000 rpm (4^oC) in the SS34 rotor for 15'
4. A pellet should be visible. If so, discard the supernatant. Dissolve the pellet in 15ml of 10 mM Tris pH8, 1 mM EDTA, 0.1 M NaCl. The pellet will be slow to dissolve but can be facilitated by triterating with a pipette and heating the sample at 37^oC.
If a PEG precipitate is not visible, the DNA may have failed to precipitate. The DNA concentration needs to be above 100 ug/ml in order to PEG precipitate efficiently. DNA can be recovered from the PEG supernatant by adding 2 volumes of ethanol, placing on ice for 20 minutes, centrifuging at 15,000 rpm in SS34 and discarding the supernatant. The pellet should be dissolved in 1 ml of TE and checked for the presence of plasmid.

Spermine precipitation.

1. Add 750ul 100mM spermine (stocks stored at -20^oC), mix thoroughly by inverting. Incubate sample on ice for 30'.
2. Collect DNA by centrifuging at 15,000 (4^oC) for 15' in the SS34 rotor. The pellet will be smaller than the PEG pellet but still should be visible. Remove as much of the supernatant as possible. (If no pellet is visible, either the DNA did not precipitate or the DNA has been lost at an earlier step. Nucleic acid can be recovered from the spermine supernatant by adding 1/10 volume of 3M NaAcetate and 2.5 volumes of ethanol. A portion of the ethanol precipitated material can be checked on a minigel for the presence of plasmid.)
3. Dissolve spermine precipitate in 5 ml of 0.6 M NaAcetate pH 6. The pellet will be slow to dissolve but can be facilitated by triterating with a pipette and heating the sample at 37^oC.
4. After the pellet has dissolved, add 5 ml of dH₂O and 20 ml of ethanol. Mix thoroughly by capping and inverting the tube several times. Let the sample stand at room temperature for a 5'. A good preparation should yield a large white fluffy precipitate shortly after mixing. If this happens, proceed with the next step. If it does not happen, you do not have very much DNA but you may be able to recover some DNA by incubating the sample on ice for 15' before proceeding with the next step.
5. Centrifuge the sample at 10,000 rpm (4^oC) for 15' in the SS34.
6. Discard supernatant and repeat the ethanol precipitation by first dissolving the DNA in 5 ml of 0.6 M NaAcetate pH 6 and proceding as described above.  A second round of ethanol precipitation seems to be necessary to eliminate spermine that tends to carry over with the first round of ethanol precipitation. If the second round is omitted, the residual spermine will cause the DNA in the final DNA stock to precipitate over long periods of storage.
7. Add 5 ml of 75% ethanol (-20^oC) to the precipitate, swirl, centrifuge at 10,000 rpm (4^oC) for 5' in the SS34.
8. Discard supernatant and allow the pellet to air dry or place it in a vacuum jar.
9. Dissolve DNA in 1 or 2 ml of 10 mM Tris-Cl pH8/ 1 mM EDTA. Store at 4^oC. From 1 liter of cells, you should obtain at least 1mg of DNA and probably several times that amount.