I've tried transformation of a 1.7 kb gene with DH5 alpha strain of competent cells and also used PuC19 as a control. After selecting colonies and broth-culturing overnight, I isolated plasmid. Only the Puc19 is visible after gel electrophoresis. Even If the transformation of my target insert is not successful, the Plasmid of the competent cells (without insert) should have been visible. What can be the possible reasons for this failure? Thanks in advance.
Maybe you should check the selection plate. Are you using the right antibiotic at the right concentration?
Yes I'm using Amp @ 50ug/ml plates and earlier I got good plasmid results and using primers corresponding to my insert, I amplified that Plasmid but I was getting the amplicon of lower size than the insert (control). How is it possible even after using the same primers?
Just do it once again. You have to get a plasmid in each lane. Make sure that your liquid contains AMP as well. Do a mock transformation without plasmid - if this control is empty, your preps have to contain a plasmid.
Possible reason is that you are using low antibiotic concentration, optimum antibiotic concentration for Amp is 100ug/ml. And be careful to use freshly prepared agar plates as amp have very short half life at 37C.
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