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% 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 4o
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
25% potassium acetate 250 g potassium acetate
add 150 ml glacial acetic acid and bring up to 1 liter
store at 4o
40% PEG 8,000 40 g PEG 8,000
up to 100 ml with Q
store at room temperature
Spin down 5 ml of a overnight culture in 3 eppendorf tubes at 14K and pool the samples by resuspending in 200ml Solution I containing 1 mg/ml Lysozyme (Sigma #L6876) on ice.
Immediately add 400ml Solution II, followed by 300 ml Solution III and spin at 14K for 5-10'.
Following the spin, remove 600-700ml of the supernatant and add 500 ml phenol/chloroform; mix and spin at 14K for 5'.
Remove 600ml supernatant, top off with cold EtOH and spin 10' at room temperature.
Wash with 80% EtOH, dry and resuspend in 60ml TE plus 1 ml RNaseA (10mg/ml); incubate at 37° for 10'.
Add 50ml 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 90ml Q (for pUC ori I get approx. 0.2-0.5 mg/ml).