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| I have repeated three times this cloning and every time I get numerous colonies none of which contains any insert. Actually I am trying to clone a EcoRI cut insert into a vector that is almost 9kb. I check my clones by colony pcr from the overnight grown single colonies from the bacterial plates. This time I set up a control while performing transformation. I transformed -vector EcoRI cut and purified, I got three colonies -vector EcoRI cut, column purified and CIP treated and column purified, I got 7 colonies -vector EcoRI cut, column purified and CIP treated and column purified plus insert EcoRI and purified in the ratio of 1:4 and 1: 6. I got more than 100 colonies. Can anyone explain what went wrong? None of the colonies contained the insert when I checked them using primers specific for the insert. However, control pcr worked. Control contained the insert, EcoRI purified fragement. |
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| blund , cloning , end , fails , reason |
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