Hi, i doing my research to ligate my insert to vektor pBI121. i used 2 enzim for dygest vektor both of them is hindIII and BamHI and I continue with blunt end them both.. i do ligate for several time but i didnt find my insert on it after I ligate and culture them in plate (self ligation) and i had confrm with PCR confirmation but there's no insert on it.
i cant find my insert in PCR confirmation, how about that? is there any mistake?
Hello Agus,
First of all I have several questions that I would like to ask you.
1. How are you exactly "blunting" your plasmid and insert ? Are you filling the 5'-ends or are you removing the 3'-ends ?
2. Are you doing exactly the same protocol with your insert ?
3. (And last) Do you have an interest to keep the orientation of your insert ?
I might have some recommendations for you...
thank Brother Paulo Juarez
First of all I have several questions that I would like to ask you.
1. How are you exactly "blunting" your plasmid and insert ? Are you filling the 5'-ends or are you removing the 3'-ends ? > i blunt them all cause there's no enzym that i can use for single digestion.
2. Are you doing exactly the same protocol with your insert ? yes i am,
3. (And last) Do you have an interest to keep the orientation of your insert ? No, i just wanna check via sequensing for this.
I might have some recommendations for you...
Blunt end ligation problem, can anyone help? - ResearchGate. Available from: https://www.researchgate.net/post/Blunt_end_ligation_problem_can_anyone_help [accessed Mar 21, 2017].
thank you brother,, i need your answer for your question above, do i have a mistake?
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