greetings
im trying to ligate a 555bp insert into a 6kb vector both plasmids were digested with pci I to extract back bone n insert of interest... i did dephosphoylation of backbone, n ligated at 22 degrees celcius overnight, the next day i transformed half the ligation mix n plated...the other half i left at room temp to transform the next day incase the first didnt work, n the following day i had no colonies.. my positive control plate had a few colonies, 1 of my experimental plates (A) had four another ( B ) had alot the others non,, i took 10 from plate b and all four from a, after overnight inoculation of plate b colonies nothing had grown as the solution of LB was still clear.. i did have trouble picking colonies from plate b because they had small colonies n we use a pipette tip which i think sometimes digs the colony into the plate rather then lifting it up. the four colonies from plate a grew n i mini prepped n digested but the bands were not of correct size they were not even the sizes of a religated backbone, i dont know what thy couldve been... i transformed the remaining ligation mixture but the next day i had no colonies on any plates...how do i better my ligation protocol?... i used kapa ligase kit, i even used thermo t4 kit, i used a 1:3 ratio of vector insert.. i did have very low dna concentrations of vector n insert though.. so i also tried adding a lot more insert because the starting concentration was low.
Edited by KhaleesiDany, 23 January 2015 - 01:45 AM.