Ligation
From 2007.igem.org
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| + | '''Equipment and reagent''' | ||
| + | *T4 DNA Ligase | ||
| + | *10X ligation buffer (0.66m Tris-HCl,pH 7.6,50mM MgCl, 50mM DTT, 10mM ATP) | ||
| + | *low temperature water-bath or heating-block | ||
| + | *Gel electrophoresis apparatus | ||
| + | |||
| + | '''Method''' | ||
| + | |||
| + | 1. The volume of the ligation mixture and the DNA concentration depend on the type of ligation experiment.Use a 10μL reaction with DNA a concentration of > 100ng/μL to promote the formation of concatenamer ligation products,or a 10μL(or larger)reaction with DNA at a concentration < 10ng/μL to promote the formation of circular ligation product. | ||
| + | |||
| + | 2.Add T4 DNA ligase.Check the supplier'documentation on enzyme activity,or more generally,add 0,25 U/μg of DNA for cohesive-end ligations,and 2,5U/μg of DNA for blunt-and ligations | ||
| + | |||
| + | 3.Incubate the reaction mixture 3,5h at ambient temperature | ||
| + | |||
| + | 4.Analyse for correct and complete ligation by gel electrophoresis using unligated material as a suitable control or also by transformation of E.coli cells if appropriate. | ||
Latest revision as of 15:17, 20 September 2007
Equipment and reagent
- T4 DNA Ligase
- 10X ligation buffer (0.66m Tris-HCl,pH 7.6,50mM MgCl, 50mM DTT, 10mM ATP)
- low temperature water-bath or heating-block
- Gel electrophoresis apparatus
Method
1. The volume of the ligation mixture and the DNA concentration depend on the type of ligation experiment.Use a 10μL reaction with DNA a concentration of > 100ng/μL to promote the formation of concatenamer ligation products,or a 10μL(or larger)reaction with DNA at a concentration < 10ng/μL to promote the formation of circular ligation product.
2.Add T4 DNA ligase.Check the supplier'documentation on enzyme activity,or more generally,add 0,25 U/μg of DNA for cohesive-end ligations,and 2,5U/μg of DNA for blunt-and ligations
3.Incubate the reaction mixture 3,5h at ambient temperature
4.Analyse for correct and complete ligation by gel electrophoresis using unligated material as a suitable control or also by transformation of E.coli cells if appropriate.
