I do not detect my insert in my colonies via colony pcr. What is the reason? I wanted to clone a shRNA in pRNAi. I digested my plasmid & pcr product of shRNA with the same RE. Then ligate them together . I tested 3 ratio of ins/vec 1:1, 5:1 and 10:1. then transformed them to ecoli.(a 65 bp has to exit from PRNAi after digestion and my 80bp shRNA replace to it). Now I have 3 plate each of them contain almost 50 colonies. Until now, I have tested 50 of them with colony pcr and all of my tested colonies were negative for my insert. Is it possible? Maybe all of the colonies have self ligation? Can any one help?
thanks sam
If I want to replace my method on the basis of your view, what can I do for extraction & preparing of my vector?
I set up my mastermix and just have negative control.I also tested my colonies with digestion, but untill now I have not seen the 80bp band( insert) on the 4% gel electrophoresis.I think the majority of my problem linked to the digestion & purifing of the pRNAi.what can I do?pls help me.
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