I have been trying insert my reporter construct (1.8kb - taken from another plasmid) into my lentiviral vector (9.6kb). They have compatible restriction sites - NheI and EcoRI. I digested the plasmid and insert with the enzymes; gel purified my vector backbone and insert and set up the ligation reaction.
Result of the plates are:
A - ligation reaction - approx 25 colonies. (used 20ul of the ligated mix)
B - vector backbone only - approx 10 colonies
C - insert only - approx 10 colonies
D - no ligase - several colonies
E - EcoRI linearized vector - several colonies
F - EcoRI linearized plasmid containing the insert - no colonies
G - only competent cells - no colonies
I don't understand why i have colonies in my insert only plate. the insert doesn't even have Amp resistance gene. Could there have been some contamination with the vector backbone? Why do i have colonies in the vector backbone only control? does the T4 ligase also ligate uncomplimentary ends in a plasmid??
You can get a tiny bit of cross contamination without noticing, maybe from your pipets or maybe when you purified your fragments. It is rare you get the background to zero. But go ahead and screen the colonies from your ligation mix and maybe some will be correct.
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