Dephosphorylation Prior to Cloning
Application Overview
Dephosphorylation is a crucial step in cloning protocols where a DNA fragment is ligated into a plasmid vector. The process aims to reduce the occurrence of "empty clones" or background, which can result from the vector religating on itself. Shrimp Alkaline Phosphatase (SAP) offers a convenient and effective solution for this, streamlining the dephosphorylation process and enhancing cloning efficiency.
Application Overview
Dephosphorylation is a crucial step in cloning protocols where a DNA fragment is ligated into a plasmid vector. The process aims to reduce the occurrence of "empty clones" or background, which can result from the vector religating on itself. Shrimp Alkaline Phosphatase (SAP) offers a convenient and effective solution for this, streamlining the dephosphorylation process and enhancing cloning efficiency.
THE PROBLEM These ENZYMEs SOLVE
In cloning, especially when a single restriction enzyme is used to cut the vector, there's a high likelihood of the vector religating back onto itself rather than incorporating the desired DNA fragment. This results in a significant number of "empty clones," which are essentially background noise in the cloning process. Traditional phosphatase treatments to reduce this background can be cumbersome and error-prone.
THE PROBLEM These ENZYME SOLVES
In cloning, especially when a single restriction enzyme is used to cut the vector, there's a high likelihood of the vector religating back onto itself rather than incorporating the desired DNA fragment. This results in a significant number of "empty clones," which are essentially background noise in the cloning process. Traditional phosphatase treatments to reduce this background can be cumbersome and error-prone.
THE PROBLEM These ENZYMes SOLVE
In cloning, especially when a single restriction enzyme is used to cut the vector, there's a high likelihood of the vector religating back onto itself rather than incorporating the desired DNA fragment. This results in a significant number of "empty clones," which are essentially background noise in the cloning process. Traditional phosphatase treatments to reduce this background can be cumbersome and error-prone.
The Solution
SAP provides a more convenient and reliable method for dephosphorylation. It can be completely inactivated by a simple heat step and is compatible with all buffers used for restriction enzymes. This allows SAP to be added either during or after restriction digestion. With SAP, the user can eliminate elaborate calculations and multi-step incubations, as the enzyme effectively dephosphorylates DNA in a single, straightforward incubation, reducing the background of "empty" clones by over 95%.
There are many ways to use SAP 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, SAP is present during restriction cutting, so the termini are dephosphorylated as soon as they are formed. In this protocol, the minimum effective amount of SAP is proportional to the amount of restriction enzyme added (i.e. the rate of terminus formation). In the simple sense, use at least 0.1 U SAP per Unit restriction enzyme, and proceed to complete cutting. The amount of restriction enzyme may differ from the protocol below, please use amounts recommended by the supplier.
- 1 µg plasmid
- 5 Unit restriction enzyme
- 5 µl 10x restriction enzyme buffer
- 1 Unit SAP
- 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 SAP. When restriction cutting is complete, simply add 5 U SAP 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.
Application Background
