CIP Treatment

From 2007.igem.org

CIP treatment is done to phosphatase the vector used for plasmid ligations. This is done to reduce the self ligation of a vector digested with enzyme(s) creating compatible sticky ends and hence enhance the S/N ratio of transformations.

Use 10 units of CIP per 1µg of DNA (over digesting by factor of X)

Calculate volumes

DNA µg = DNA volume * concentration

Enzyme volume = Enzyme unit/µl* # units = X [µl]

Buffer is dilution factor x dilution of the total volume.

[i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] Order of filling

DNA

Water

Buffer

CIP

Incubate for 1 hours at the specified temperature for the enzyme (37C). Keep the buffer on ice and the CIP in the benchtop coolers when on the bench.