Amplification of Low Copy Plasmid

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Fizz
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Amplification of Low Copy Plasmid

Hi !

I was wondering if someone could guide me towards a working method to isolate a low copy plasmid? I was leaning towards the chloramphenicol amplification method. If someone has some experience using that and is willing to share that information it would be greatly appreciated.

cfish
cfish's picture
Chloramphenicol amplification

Chloramphenicol amplification is a common method for isolating low copy plasmids. Or increase number of cells for plasmid isolation.

Jason King
Jason King's picture
This might be a bit of an old

This might be a bit of an old wive's tale, but I've heard it said that simply placing an overnight culture in the coldroom stops the E.Coli dividing but allows plasmid replication to continue (to some extent).

Irrespective of whether you do this or use chloramphenicol, I've found it very useful to pre-precipitate the DNA before loading it onto a Qiagen column. So you'd do a 500mL LB amp culture. Spin it down and do the P1, P2 and P3 steps. For this I would increase the volume of each solution to 20mL (instead of the 10mL recommended in the protocol).

Then after the high speed centrifugation, transfer the supernatant into 2 50mL tubes (30mL each). Fill up each tube with isopropanol. MIX WELL.
Centrifuge fast for 30 mins at 4 degrees to pellet the DNA.

Discard supernatant. Use a P200 to remove as much liquid as possible. Allow the pellet to dry a LITTLE (no visible liquid). Add 1mL of water to resuspend the DNA - Gently.When resuspended, add about 10 mL of QBT.

Load this on to the pre-equilibrated column and then follow the manufacturer's instructions.

This is very good for getting rid of a lot of biomass pre-column loading and allows more of the plasmid to bind efficiently.