Quint Lab:ligation (sticky ends)

{| cellspacing="2px" cellpadding="0" border="0" style="padding: 0px; width: 750px; color: #000000; background-color: #ffffff;"
 * -valign="top"
 * width=300px style="padding: 5px; background-color: #ffffff; border: 2px solid #F8B603;" |

ligation
ligation mix: incubate O/N at 16ºC.
 * 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

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