After transforming cells (see Standard Transformation Protocol) and plating cells (see
Start out with the whole cells that have been incubating. They will be in LB medium.
Spin the cells in a centrifuge for 5 minutes at 4000 rpm. After 5 minutes the cells should be pelted, if they are not spin longer.
Remove remaining liquid which is known as supernatant. Be careful not to touch the pellet of cells at the bottom.
Add 250uL of suspension buffer/RNase. Resuspend the cells in the RNase. RNase is a very dangerous chemical in the lab because it gets rid of the RNA in a cell. Be very careful about using the RNase in the lab.
Transfer the resuspended cells into a 250mL eppendof tube.
Add 250 mL of Lysis Buffer. Lysis buffer will break the cells so the contents of the cells will be free-floating in the solution.
Let them sit at room temperature for 5 minutes before preceding. The solution should become less cloudy.
Add 350uL of chilled binding buffer.
Put them on ice for 5 minutes.
Centrifuge them for 10 minutes at 13 rpm.
There will be protein at the bottom of the eppendorf tube, only remove the supernatent (liquid).
Put the supernatent in the stop of the filter and spin for 1 minute.Discard the flowthrough.
Add 500uL of wash buffer I. Centrifuge for 1 minute. Discard flowthrough.
Don't add anything, centrifuge for 1 minute to dry filter.
Put dry filter in a new eppendorf tube.
Add 50uL of Elution Buffer. Centrifuge for 1 minute, remove and discard filter.
End Result: 50uL of plasmid.