I have two linear DNA fragments that will be ligated together (each ~700 bp).
This fragment then needs to be ligated into a linearised vector (plasmid).
Would I need to perform a clean up on the first reaction, if it will be directly used for a successive ligation reaction?
It probably depends upon the restriction sites that are being used and whether you intend to separate the ligated fragment from the two input fragments. If the ligated fragment will have different ends than the input fragment you could even consider just doing a single ligation with all 3 DNA pieces.
Dear Nikki Schütte
I'm agree with Michael J. Benedik that in case of different ends, you can try to perform also a ligation with all the 3 DNA pièce and try to select the good clones using PCR coloni screening.
Moreover, you can also fuse the 2 fragment by PCR addomg an overlapping regions with the primers and afterward perform just a single ligation with the big fragment digested, since PCR when work will produce higher amount of material and in case is necessary to perform a PCR purification before a 2nd digestion step you will have an enough amount of material.
good luck
Manuele
Yes, it is recommended to perform a clean-up step after each ligation reaction, even if it will be directly used for a successive ligation reaction.
The ligation reaction may generate unwanted byproducts, such as unligated fragments or ligated fragments that contain multiple inserts or mutations. These byproducts can interfere with the subsequent ligation reaction and reduce its efficiency.
Therefore, a clean-up step, such as gel electrophoresis or column purification, can help remove the unwanted byproducts and ensure that only the desired product is used for the subsequent ligation reaction.
Additionally, some ligases may require certain buffer conditions or DNA purity levels for optimal activity, so a clean-up step can also help ensure that the reaction conditions are suitable for efficient ligation.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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