DNA Plasmid Prep.

This is a scaled up version of the Baron protocol which has been modified to achieve purity comparable to CsCl preps. I have not sorted out strain variations yet but HB101 works well.

1、Solutions

Solution I　　1% glucose 10 g glucose　　25 mM Tris pH 8.0 25 ml 1M Tris pH 8.0　　10 mM EDTA 20 ml 0.5 M EDTA pH 8.0　　up to 1 liter with Q　　store at 4 ℃

Solution II　　1% SDS 2.5 ml 20% SDS　　0.2N NaOH 1.0 ml 10N NaOH　　up to 50 ml with Q　　make fresh as needed

Solution III　　25% potassium acetate 250 g potassium acetate　　add 150 ml glacial acetic acid and bring up to 1 liter with Q　　store at 4 ℃

2、PEG 8000

40% PEG 8,000 40 g PEG 8,000　　up to 100 ml with Q　　store at room temperature

3、Procedure

• Spin down 5 ml of a overnight culture in 3 eppendorf tubes at 14K and pool the samples by resuspending in 200 ml Solution I containing 1 mg/ml Lysozyme (Sigma #L6876) on ice.　　• Immediately add 400 ml Solution II, followed by 300 m l Solution III and spin at 14K for 5-10'. 　　• Following the spin, remove 600-700 ml of the supernatant and add 500 ml phenol/chloroform; mix and spin at 14K for 5'. 　　• Remove 600 ml supernatant, top off with cold EtOH and spin 10' at room temperature. 　　• Wash with 80% EtOH, dry and resuspend in 60 ml TE plus 1 ml RNaseA (10mg/ml); incubate at 37 ℃ for 10'. 　　• Add 50 ml phenol/chloroform and spin for 2', remove 50 ml supernatant and add 10 ml 5 M NaCl plus 21 ml 40% PEG 8000. 　　• Incubate on ice 5', spin 5' at room temperature and resuspend in 90 ml Q (for pUC ori I get approx. 0.2-0.5 mg/ ml).