"
CIP Treatment
From 2008.igem.org
(New page: ==CIP Protocol==) |
|||
| Line 1: | Line 1: | ||
| - | ==CIP Protocol== | + | ==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 enhance the S/N 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. | ||
Revision as of 23:50, 28 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 enhance the S/N 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.
