plasmid DNA maxi prep

2 posts / 0 new
Last post
bsengez
bsengez's picture
plasmid DNA maxi prep

Hello everybody,
I have Invitrogen Purelink maxi prep kit and I'm using high copy number plasmids as -80 stocks. Is there anyone using this kit? By the way, with how much culture are you starting?

Jason King
Jason King's picture
I've never used the

I've never used the Invitrogen kit but it will probably say in the instructions that you should try to reduce the amount of biomass for high copy plasmids. So if the columns are supposed to bind 500 micrograms you should use about 200mL of culture.

If you need to purify more than 500g and want to save a little money, you can load and elute twice. If you want to do this, then I would suggest the following:

Do a 400-500 mL overnight culture. Spin down the bugs, resuspend in the first solution. Lyse the cells in the second solution then neutralize using the third solution. Pellet the precipitated cell debris at high speed and transfer the supernatant.

Normally you would load this on to the column. Instead of doing this, you can transfer the supernatant to 50 mL Falcon tubes and add 0.7 volumes of RT isopropanol, mix well and centrifuge. Remove as much liquid as possible (using a P200 pipette) and allow the pellet to air dry a LITTLE (NOT TOO MUCH, since this tends to prevent the re-solution of the DNA). Add about 1mL of dH2O to disolve the DNA - it should go into solution easily and "fizz around" in the water like a piece of lithium or calcium metal ;)

When the DNA has disolved, dilute it in the buffer used to equilibrate the column. Equilibrate the column and then load it with half of the diluted DNA. Then follow the protocol for washing and eluting. Then wash the column through with wash buffer and then re-equliibrate the column and do the second loading etc.

The DNA "pre-precipitation" gets rid of a lot of the protein so that the sample that is loaded onto the column is cleaner. This should allow a bit more DNA to bind too.

PM