Editing PEG Maxiprep (section)

== 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