Ligation (McGill)
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[[McGill_University_2006|Home]] <br> | [[McGill_University_2006|Home]] <br> | ||
[[Protocols|Protocols]] | [[Protocols|Protocols]] | ||
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| + | For a ligation, the optimal vector to insert ratio is a 1:3 molar ratio. To calculate this, one must consider the original concentrations of the DNA samples and the sizes of the DNA fragments in bp. The total amount of DNA in the ligation mixture should be between 100 and 200 ng. | ||
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| + | In a small tube, mix the following components, being sure to add the enzyme last. | ||
| + | * x volume of insert | ||
| + | * y volume of vector | ||
| + | * 20-4-x-y uL of dH2O | ||
| + | * 2 uL of ligation buffer | ||
| + | * 2 uL of T4 DNA ligase | ||
| + | |||
| + | Incubate on benchtop for 1 to 2 hours, or overnight at 16 C. | ||
Latest revision as of 18:53, 13 July 2006
For a ligation, the optimal vector to insert ratio is a 1:3 molar ratio. To calculate this, one must consider the original concentrations of the DNA samples and the sizes of the DNA fragments in bp. The total amount of DNA in the ligation mixture should be between 100 and 200 ng.
In a small tube, mix the following components, being sure to add the enzyme last.
- x volume of insert
- y volume of vector
- 20-4-x-y uL of dH2O
- 2 uL of ligation buffer
- 2 uL of T4 DNA ligase
Incubate on benchtop for 1 to 2 hours, or overnight at 16 C.
