CIP Treatment

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==CIP Treatment Protocol==
==CIP Treatment Protocol==
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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.
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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 enhancing the Signal/Noise ratio of transformations.
1.  Use 10 units of CIP per 1µg of DNA (over digesting by factor of  X)
1.  Use 10 units of CIP per 1µg of DNA (over digesting by factor of  X)

Latest revision as of 00:58, 30 October 2008

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CIP Treatment Protocol

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 enhancing the Signal/Noise ratio of transformations.

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

2. 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]


3. Order of filling

• DNA

• Water

• Buffer

• CIP

4. Incubate for 3 hours at the specified temperature for the enzyme (37C).

5. Keep the buffer on ice and the CIP in the benchtop coolers when on the bench.