PEG Maxiprep

Large scale DNA Purification by PEG precipitation

Perparation of Crude Lysate by Alkaline Lysis

1. Inoculate 500mL LB media with and grow O/N at 37oC until culture is saturated (OD600~4.0)

2. Collect cells by centrifuging 10 min at 6000 x g. (~6000 rpm in GSA rotor)

If necessary, pellets can be stored frozen indefinitely at -20oC or -70oC

3. Resuspend pellet in 4 mL glucose/Tris/EDTA solution and transfer to oakridge tube

4. Add 1 mL of 25mg/mL hen egg white lysozyme to cells and allow to stand at room temperature for 10 minutes.

Lysozyme aids in destruction of bacterial cell walls and can be omitted. Omission of lysozyme reduces plasmid recovery by 5 – 10%.

5. Add 10 mL freshly prepared 0.2 M NaOH/1% SDS and mix by stirring gently with a pipet until solution becomes clear. Let stand on ice for 10 minutes

Solution will become very viscous

6. Add 7.5 mL of 3 M potassium acetate solution and stir until precipitate forms. Let stand on ice for 10 minutes

7. Centrifuge 10 minutes at 20,000 x g (13,000 rpm in sorvall SS-34)

Pellet will contain mostly chromosomal DNA, SDS-protein complexes and other cellular debris. Plasmid DNA remains in supernatant. Addition of ~0.5 mL chloroform before centrifugation will reduce floating material.

8. Decant supernatant into clean centrifuge tube. Pour through several layers of cheesecloth if floating material is visible

If supernatant is cloudy or contains floating material, repeat centrifucation before continuing.

9. Add 0.6 volumes isopropanol, mix by inversion, and let stand at RT for 5-10 minutes

10. Pellet nucleic acids by centrifuging 10 min at 15,000 x g (11,500 rpm in SS-34 rotor)

11. Wash pellet with 2 mL of 70% ethanol. Aspirate ethanol and dry pellet. The pellet can be stored indefinitely at 4oC

Remove contaminants from crude lysate pellet

1. Resuspend pellet in 1 mL glucose/Tris/EDTA solution

2. Add RNAse to final concentration of 20 µg/mL and incubate 20 minutes at 37oC

3. Add 2 mL freshly prepared 0.2M NaOH/1% SDS, mix by inverting the tube and let stand at room temperature for 5 – 10 minutes

4. Add 1.5 mL of 3M potassium acetate solution, mix by inverting and let stand 5 – 10 min

5. Centrifuge for 10 min at 20,000 x g (11,000 rpn in SS-34 rotor)

6. Transfer the supernatant to clean tube

White precipitate is SDS-potassium complex which includes chromosomal DNA remaining from crude lysate.

7. Phenol/Chloroform extract and then extract with 24:1 chloroform/isoamyl alcohol

8. Add ¼ volume of 10M ammonium acetate (2M final concentration) to aqueous phase and mix

9. Add 2 volumes of 100% ethanol and place tube on dry ice for 10 minutes

10. Pellet DNA by centrifuging 10 min at 10,000 x g (8000 rpm in SS-34 rotor)

11. Wash pellet with 70% ethanol and air dry

PEG precipitate plasmid

1. Resuspend pellet in 2 mL TE and add 0.8 mL PEG solution. Incubate for 1 – 15 hrs at 4oC

The percentage of plasmid DNA recovered by PEG precipitation increases with time of incubation. Usually ≥50% can be recovered after 1 hr. Incubation of ≥12 hrs will result in 100% recovery of plasmid DNA

2. Recover plasmid DNA by centrifuging 20 min at 10,000 x g in sorvall or benchtop (8000 rpm in SS-34)

Pellet will not be seen as clearly as an ethanol pellet. Proceed even if you don’t see a clear pellet.

3. Resuspend DNA in 1 mL TE and ethanol precipitate using 3M sodium acetate [pH 5.5]

Pellet can be stored indefinitely at 4oC

PEG solution

30% (w/v) PEG 8000

1.6 M NaCl

Store indefinitely at 4o C

Potassium acetate solution (3M), pH ~5.5

294 g potassium acetate (3M final)

50 mL 90% formic acid (1.18 M final)

Bring to 1 liter with H2O

Store indefinitely at Room temperature

Glucose/Tris/EDTA (GTE) solution

50 mM glucose

25 mM Tris-Cl [pH 8.0]

10 mM EDTA

Autoclave and store at 4o C