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Protocol D

Protocol D.1

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.

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 4o

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 4o

PEG 8000

40% PEG 8,000 40 g PEG 8,000

up to 100 ml with Q

store at room temperature

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 ml 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).