Dephosphorylation prior to cloning
In cloning protocols, a DNA fragment is ligated into a plasmid vector. If the vector is cut with a single restriction enzyme, the chances are much higher that the vector re-ligates back on itself rather than on an added DNA fragment. This results in a high fraction of “empty clones”, or background.
Alkaline phosphatases are routinely used to reduce the background from empty, re-ligated vectors during cloning of DNA fragments, since dephosphorylated DNA termini cannot be ligated by DNA ligase. The phosphatase treatment will effectively reduce the background of “empty” clones by >95%. However, cloning procedures and also phosphatase treatment may be cumbersome and error-prone.
Cryophile Shrimp Alkaline Phosphatase offers greater convenience for this procedure, since the enzyme activity may be completely removed by a simple heat inactivation step. Cryophile Shrimp Alkaline Phosphatase is active in all buffers used for restriction enzymes, so can be added either during restriction digestion, or directly after. With Cryophile Shrimp Alkaline Phosphatase the user can forget elaborate calculations and multi-step incubations, because the enzyme completely dephosphorylate DNA during one, simple, incubation.
There are many ways to use Cryophile Shrimp Alkaline Phosphatase for vector dephosphorylation, and we recommend two protocols below. These protocols apply to all types of DNA termini: 3’-protruding, blunt, or 5’-protruding.
Protocol including restriction cutting
In this protocol, Cryophile Shrimp Alkaline Phosphatase is present during restriction cutting, so the termini are dephosphorylated as soon as they are formed. In this protocol, the minimum effective amount of Cryophile Shrimp Alkaline Phosphatase is proportional to the amount of restriction enzyme added (i.e. the rate of terminus formation). In the simple sense, use at least 0.1U of Cryophile Alkaline Phosphatase per unit of restriction enzyme, and proceed to complete cutting. The amount of restriction enzyme may differ from the protocol below; please use amounts recommended.
- 1 µg plasmid
- 5 Unit restriction enzyme
- 5 µl 10x restriction enzyme buffer
- 1 Unit Cryophile Shrimp Alkaline Phosphatase
- dH2O to 50 µl
Incubate at 37°C for 1 hour, inactivate as recommended for the restriction enzyme used. Proceed to ligation protocol.
Quick dephosphorylation of cut plasmid
Efficient dephosphorylation can be achieved in short time using high amounts of Cryophile Shrimp Alkaline Phosphatase. When restriction cutting is complete, simply add 5 U Cryophile Shrimp Alkaline Phosphatase per µg vector to your restriction mix and incubate for further 5 min at 37°C. Inactivate as recommended for the restriction enzyme. Proceed to ligation protocol.
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