Hi there,
There are five DNA fragments(5’ phosphorylated) in which some sequences between them are same and after mixing together they may assembled together as they have similar sequences, it shows in the figure.
My objective is ligation confirmation
Below is the current process I’m doing:
1 Ligation, 2 Gel electrophoresis, 3 Gel Purification, 4 PCR, 5 Gel Electrophoresis
I think with above process though we purified the Gel but there would be some possibility of being hybridized DNA before PCR
Is there any way to confirm or separate ligation and hybridization from each other?
Thank you.
Dear Karimi,
You can try some traditional microbiological screening methods, such as blue white screening, etc. to confirm ligation and hybridization. For molecular methods, you can try southern hybridization method for the same. In this case, do use the fragments from all samples as well as the vector. Then add the supposed ligated system. Compare your results with the ladder / marker, to identify the ligation. You can also go for sequencing.
Hi, karimi!
I cannot find the way to solve your question right now. But I would like also to recommend southern hybrid method. And I will try to find better way.
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