April 26 1990

Matthew S. Holt

Purpose:

The recovery of highly purified recombinant plasmid DNA using a CsCl2 gradient. Milligram yields are expected.

Special Reagents:

• Plasmid Prep Solution 1 (PP1) stored at 4 degrees C
• Plasmid Prep Solution 2 (PP2) stored at 4 degrees C - shelf life of 3 months
• Plasmid Prep Solution 3 (PP3) stored at 4 degrees C
• Lysozyme cocktail - must be made fresh each time
• 6.2M CsCl2 in 1X TE

Special Equipment:

• Beckman Ultracentrifuge
• Beckman SW28 rotor and centrifuge tubes (cat # 344058)
• Beckman VTi80 or VTi65 rotor, spacers, torque wrench, hex nuts
• Beckman quick-seal tubes (cat # 342412)
• Heat sealer for quick-seal tubes

Time required:

• Day 1 - 16-20 hours to grow cultures
• Day 2 - 3 hours to the PEG precipitation. PEG precipitation occurs overnight.
• Day 3 - 1 hour to prepare ultracentrifuge tubes 2 four hour spins
• Day 4 - 2 hours to clean precipitate and resuspend DNA

Procedure:

Day 1

1. Grow an overnight culture of the desired plasmid-containing bacteria by inoculating 350 ml 1X LBM+ antibiotic (for ampicillin use a final concentration of 100 ug/ml) from one single isolated colony. Chloramphenicol amplification is no longer necessary with the current vector plasmids (see Plasmid Subcloning). Incubate in shaker incubator at 37 degrees C for 16-20 hours until the culture is dense (do not grow longer than 20 hours). If you desire more than one culture for any given plasmid inoculate 15 ml of 1X LBM+ antibiotic incubate for 2 hours at 37 degrees C in a shaking incubator and divide among the desired number of 350 ml cultures (six cultures is convenient because that is the capacity for the SW28 rotor).

1. Pellet the cells in large centrifuge bottles (500 ml capacity) in the Beckman J-6 centrifuge using the JA-10 rotor at 10000 rpm for 10 minutes 4 degrees C. Decant the supernatant and place pellets on ice. Steps 2 through 4 below can be done in the centrifuge bottle.

2. Resuspend by pipetting pellet in 10 ml 4 degrees C PP1solution. Solution should be of uniform consistency before continuing because samples will become quite viscous in the subsequent steps and pipetting becomes impossible. Hold on ice for 10-15 minutes. Samples should start becoming slightly viscous.

3. Add 2 ml of 0.5M EDTA pH 8.0 and 2 ml of Lysozyme cocktail. Swirl and hold on ice for 15-30 minutes until samples are noticeably more viscous.

4. Add 16 ml of 4 degrees C PP2 solution swirl to mix and incubate on ice for 15-30 minutes. You should notice immediate increase in viscosity. The more viscous the solution the higher the yields. Transfer solutions to the open top Beckman tubes for the SW28 rotor.

5. Pellet the bacterial debris at 25000 rpm 30 minutes 4 degrees C using the SW28 rotor in the Beckman ultracentrifuge. The tubes must be balanced to +/- 0.1 g. NOTE: The ultracentrifuge requires for both Acceleration and Deceleration settings of "1" when using swinging bucket rotors.

6. Decant supernatant (approximately 25 ml of a moderately viscous solution) into a clean 50 ml conical centrifuge tube. Add one volume of PP3 solution (bring up to 50 ml). Place on ice for a minimum of 4 hours. Yields will be larger if the PEG precipitation is carried out overnight (14 hours). For cosmids this step must be at least 14 hours (see cosmid prep protocol).

1. Pellet the DNA: Pour half of the PEG solution into a sterile Oakridge tube and spin in the Sorval RC-5B centrifuge using the SS34 rotor at 10000 rpm for 10 minutes 4 degrees C. Decant the supernatant and repeat with the other half of the PEG solution.

   NOTE: The next spin will require the temperature in the RC-5B to be above 15 degrees C so change the temperature setting on the centrifuge to 20 degrees C and leave the door open. You will also need the SM-34 rotor to be above 15 degrees C.

2. Resuspend the pellet in 5 ml of 1X TE pH 8.0 by pipetting the TE into the pellet. The resulting solution must be of uniform consistency (no lumps). It is sometimes helpful to let the solution sit 10 minutes because the lumps will tend to dissolve. While solution is sitting measure 5.2 g of CsCl2 for each sample and place in labeled 15 ml "snap-cap" culture tubes. Also add 500 ul ethidium bromide (10 mg/ml) to each tube.

3. When the pellets are well resuspended add to the CsCl2 tubes and vortex to dissolve the CsCl2 (cosmids should not be vortexed). Do a pre- clearing spin in the Sorval RC-5B centrifuge using the SM-34 rotor at 10000 rpm for 10 minutes 20 degrees C (temperature is critical here because CsCl2 will precipitate out of solution below 15 degrees C). This spin will remove debris by forcing out anything too small or too large for the gradient and result in substantially cleaner samples for the VTi80 spins. Samples should have a small pellet on the bottom and a film layer sitting on the top after this spin. If there is a large pellet on the bottom chances are this is CsCl2. If this happens warm the samples remix and repeat this step.

4. Using a sterile pasteur pipet transfer the supernatants (avoiding the debris on top and bottom) to labeled Beckman quick-seal tubes using another sterile pasteur pipet as a funnel. There will be enough solution to fill approximately 1.2 of these quick seal tubes. For plasmids one tube is generally sufficient and the rest can be discarded. Dry the neck of each quick-seal tube with a kimwipe and balance them to +/- 0.1 g. Make a balance tube for an odd number of samples.

5. Place metal caps on the quick-seal tubes and heat seal (heat sealer requires 10 minutes to warm up). It is critical to have a good seal and advisable to practice sealing first on tubes filled only with water. Check each seal by pointing the top of the tube into the sink and applying pressure.

6. Place each tube and its counterbalance tube in the VTi80 rotor. Place a metal spacer on top of each tube. Coat the threads of each hex nut with vacuum grease and place on top of each spacer. Tighten each hex nut to 120 lbs. with the torque wrench. Place the rotor in the ultracentrifuge and spin at 75000 rpm for 4 hours 20 degrees C Acc. 1 Dec. 9 (the tubes spin vertically in the rotor and the gradient makes a 90 degrees flip while decelerating so it is necessary to decelerate as slowly as possible using a setting of 9). Reduce the speed of the run to 60000 rpm for spins longer than 4 hours (such as an overnight spin).

7. Carefully remove the rotor from the centrifuge (avoid disrupting the gradient). Remove the hex nuts with the torque wrench and remove spacers and tubes with a pair of forceps. Place the tubes on white "finger" rack used to pull the plasmid bands. There will be two distinct red bands the top one is chromosomal DNA and the bottom one is plasmid DNA. Use the hand- held UV source to find the bands if the yield is low. Remove the bands as quickly as possible because the DNA will diffuse.

8. With a syringe and an 18 gauge or larger needle make a vent in the tube by puncturing the tube at the top. With this same needle puncture the tube about 1/2 cm above the top (chromosomal) band angle the needle down and slowly draw off the band. When finished leave the syringe and needle hanging in the tube to prevent leakage. Take a fresh syringe and needle puncture the tube about 1 cm below the plasmid band angle the needle up and slowly draw off the plasmid DNA band collecting anywhere from 700 ul to 1.5 ml. When this syringe is removed the remaining solution will drain out of the tube so have an ethidium bromide waste container ready to put the tube into when finished.

9. Because the syringe can shear the DNA remove the needle from the syringe and transfer the plasmid DNA to a fresh quick-seal tube (using a sterile pasteur pipet as a funnel as before). Fill each tube with 6.2 M CsCl2. Dry the neck of each tube balance to +/- 0.1 g and repeat steps 5 6 7 and 8. Only one band should be visible after the second spin. If two bands are visible a third spin is necessary.

Day 4

1. Place DNA from the second spin in a 15 ml snap-cap culture tube (approximately 1 ml). Mark the volume level on the tube because isopropanol extracts water and it is necessary to replenish the water between each extraction to prevent the CsCl2 from precipitating. Add 1 ml of isopropanol (at room temperature) and gently mix then allow the phases to separate. Isopropanol extracts the ethidium bromide and will be the top phase. Use a pipetman to remove the isopropanol/ethidium bromide (phases will separate in the pipet tip). Replenish the DNA volume to the mark with autoclaved water and repeat at least twice until all trace of red coloring is gone. Ethidium bromide can inhibit restriction enzyme digestion.

2. Add 2 volumes of autoclaved dH2O (i.e. bring 1 ml up to 3 ml by adding 2 ml dH2O). Add 2-3 volumes of -20 degrees C 100% EtOH (i.e. add 8- 9 ml EtOH to 3 ml). If the yield is good a pellet will be visible after inverting several times to mix. For a visible pellet spin sample immediately in Sorval RC-5B centrifuge using the SM-34 rotor at 7000 rpm for 15 minutes 4 degrees C. If the yield is low i.e. no visible pellet place the tube at -20 degrees C for at least one hour then spin as above.

3. Decant the supernatant and rinse briefly with 70% EtOH; decant the EtOH wash. It isn't necessary to respin unless the pellet is dislodged.

4. Speed vac or air dry the pellets until all traces of EtOH have vanished. Resuspend the DNA in 50-100 ul (more for larger pellets) quantitate on the spectrophotometer and verify by cutting with an appropriate restriction enzyme. Run uncut and cut plasmid DNA side by side with standards on the gel to evaluate.

Solutions:

• PP1:

   	25% sucrose 	50 mM Tris*HCL, pH 8.0 	1 mM EDTA, pH 8.0  

• PP2:

    	0.1% Triton X-100 	50 mM Tris*HCL, pH 8.0 	60 mM EDTA, pH 8.0      Shelf life of 3 months  

• PP3:

    	30% polyethelyne glycol (PEG f.wt.8000) 	1.5 M NaCl  

• Lysozyme Cocktail:

    	prepare 2 ml for each sample 	made fresh each time 	3.0 ml 1 M Tris pH 8.0 	9.0 ml sterile ddH2O  	60 mg Lysozyme 	12 ml - enough for 6 samples.  

• 6.2M CsCl2:

    	Dissolve 42 grams of CsCl2 in 40 ml  	sterile 1X TE pH 8.0  

  Safety Considerations:

  Ethidium bromide is a powerful mutagen and moderately toxic. Gloves should be worn when handling. Ethidium bromide waste is contained and disposed of through W.U.M.S. Hazardous Waste Management Office. UV can cause severe burns. Always wear eye protection.

  References:

  Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp. 1.42-1.46.

  ---