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CIP Treatment
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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 | + | 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) | ||
Revision as of 01:09, 29 October 2008
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.
