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ligation
ligation mix:
- x µl digested vector
- y µl PCR fragment or digested insert
- 1 µl l0x ligase buffer
- 1 µl T4 DNA ligase [3 Weiss U/µl]
- up to 10 µl dH2O
incubate O/N at 16ºC.
in most cases, a 1:1 or a 1:3 ratio work best
formular:
[(ng vector) x (kb size of insert)] / (kb size of vector) x [molar ratio of (insert/vector)] = ng insert
example:
500bp insert to be ligated with 100ng of 3.0kb vector in a 3:1 ratio
→ [(100ng vector) x (0.5 kb of insert)] / [3.0 kb vector x (3/1)] = 50 ng insert
or simply use the vector:insert ratio calculator
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