(KA Longo, 1/01)
* The amount of insert DNA used will depend on the relative size of the insert compared to the size of the vector. A molar ratio between insert and vector should be calculated. Typically, the insert/vector ratio for sticky-end ligations ranges between 1:1 and 3:1. For blunt-ended ligations, the insert/vector ratio should be at least 10:1. The number of moles for vector and insert can be calculated as follows: (g DNA) ------------------------------------------ [(# of base pairs DNA) x (660 daltons/bp)] You can use the following equality to figure out the amount of insert DNA needed for a known amount of vector in a blunt-end ligation: (g vector DNA) (g insert DNA) ----------------------------------------- x 10 = ----------------------------------------- [(# of bp vector DNA) x (660 daltons/bp)] [(# of bp insert DNA) x (660 daltons/bp)] Or: 10 x (g vector DNA) x (# of bp insert DNA) g insert DNA = ---------------------------------------------------------- (# of bp vector DNA) Note that any desired ratio can be substituted for the number 10 in the above equation, depending on the type of ligation you are performing. |
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