CIP Treatment

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==CIP Protocol==
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==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 enhancing the Signal/Noise ratio of transformations.
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1.  Use 10 units of CIP per 1µg of DNA (over digesting by factor of  X)
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2. Calculate volumes
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DNA µg = DNA volume * concentration
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Enzyme volume = Enzyme unit/µl* # units = X [µl]
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Buffer is dilution factor x dilution of the total volume.
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[i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume]
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3. Order of filling
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• DNA
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• Water
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• Buffer
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• CIP
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4. Incubate for 3 hours at the specified temperature for the enzyme (37C).
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5. Keep the buffer on ice and the CIP in the benchtop coolers when on the bench.

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.