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Protocol C

Protocol C.1

CsCl Prep of Plasmid DNA

This is a standard large scale prep. for plasmid DNA which gives a yield of 0.5 - 1.0 mg. I have made some minor changes to the MHB protocol.

Solutions

Solution I, II, III from protocol D.1.

 

Tris/EDTA pH 7.5 (optional)

20 ml 1 M Tris 7.5

4 ml 0.5 M EDTA 8.0

up to 2 liters with Q

store at 4 degrees for dialysis

 

Dialysis Tubing (optional)

The tubing is stored dry at 4 degrees (Spectrapore 12,000- 14,000 molecular weight cut off)

cut a 1-2 meter segment and immerse in Q

autoclave for 10 minutes on fast exhaust

store at 4 degrees in 20% EtOH

prior to use, wash in TE

 

Procedure

 

Start a 500 ml culture with the appropriate antibiotic approximately 24 hours in advance by innoculating a single colony.

• Harvest the cells by spinning in a 500 ml bottle in the J6B at 4.2K for 20 minutes. Resuspend the pellet gently in 30 ml Solution I with 1 mg/ml lysozyme (Sigma #L6876).

• Transfer immediately to a 250 ml bottle on ice and add 60 ml Solution II, followed by 45 ml Solution III. Immediately spin in the Sorvall GSA rotor at 10K for 10'.

• Transfer the supernatant to a clean 250 ml bottle and spin for 10' at 10K (optional if there is still some precipitate after the first spin). Transfer the supernatant from this spin to another 250 ml bottle filtering through several layers of cheesecloth.

• Add 1 volume of isopropanol and incubate at room temperature for 5-10 minutes followed by a 10K spin for 30'.

• Thoroughly dry the pellet and resuspend in 6 ml TE. Transfer 5.5 ml to a 13 ml snap top tube and add 6.1 g CsCl plus 0.3 ml EthBr (10 mg/ml).

• Spin in the Brinkman Speedfuge for10' at 6K and transfer the supernatant to a Quick Seal Tube. Top off with heavy mineral oil, balance and seal.

• Spin at 56K rpm for at least 20 hours, remove the plasmid DNA band with a needle and extract EthBr with NaCl saturated Isopropanol. Alternately, extract with isoamy alcohol and precipitate by adding 2 volumes of 70% EtOH. Wash, dry and resuspend in 0.5 ml TE.

• If the EthBr was removed by NaCl saturated isopropanol, dialyze against 2 liters of TE at 4 degrees for 24-48 hours, and quantitate.