Hi, Ive prepared my ligation mix for sticky end ligation in the ratio of 1:3, 1:6 and 1:9 calculated using NEBio Calculator with NEB T7 DNA Ligase at 25°C for 20 minutes then transformed them in OneShot TOP10 E coli. The transformation yield colonies however upon double digestion at 37°C for 2 hours, most of them only exhibit one band at 5kb~ indicating the vector (pET28 a(+)) without any band for insert at supposedly 1.3kb. Unfortunately, for those with insert band, there is no band for vector (or maybe it is too faint?). I dont think that it is possible for only the insert to be transformed in the competent cells without the vector as insert + vector = plasmid, but why is the vector band seems undetectable? I havent digest the ligation mix yet but if the ligation is successful, what is the problem then? If they were not, please advise on how to increase the ligation efficiency. Thank you in advance :D
Hello Fatin, I used T4 DNA ligase from NEB (catalog no. M0202) for sticky end ligation successfully in past. I ligate at 16°C overnight, 1(vector):3(insert) ratio (calculator: http://www.insilico.uni-duesseldorf.de/Lig_Input.html). I dephosphorylate vector DNA and separate it by agarose gel electrophoresis prior ligation. Do not allow your vector and insert volume to be more than half volume for whole reaction. Also aliquote T4 DNA buffer as it contains ATP and is prone to degradation after freeze-thaw cycles.
Could you post a picture? Something does seem very odd if you have colonies that only have a 1.3 kb insert but no vector band (since the insert should not be able to replicate).
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