Editing PEG Maxiprep

= Large scale DNA Purification by PEG precipitation =

== Perparation of Crude Lysate by Alkaline Lysis ==

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

<br/>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

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

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

<br/>Lysozyme aids in destruction of bacterial cell walls and can be omitted. Omission of lysozyme reduces plasmid&nbsp;recovery by 5 – 10%.

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

Solution will become very viscous

<br/>6. Add 7.5 mL of 3 M potassium acetate solution and stir until precipitate forms. Let stand on&nbsp;ice for 10 minutes

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

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

<br/>8. Decant supernatant into clean centrifuge tube. Pour through several layers of cheesecloth if&nbsp;floating material is visible

<br/>If supernatant is cloudy or contains floating material, repeat centrifucation before continuing.

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

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

<br/>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 ==

<br/>1. Resuspend pellet in 1 mL glucose/Tris/EDTA solution

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

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

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

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

<br/>6. Transfer the supernatant to clean tube

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

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

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

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

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

<br/>11. Wash pellet with 70% ethanol and air dry

== <br/>PEG precipitate plasmid ==

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

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

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

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

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

<br/>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


[[Category:Protocols]]