I'm trying to ligate two adapters to a linear insert of DNA. The adapters are 59 and 69 bp long, and the insert is ~200bp. It's approximate because there are many inserts, they are the result of a golden gate assembly reaction. In the attached gel the lanes (from the left) are a 50bp ladder, the golden gate assembly, and four ligations where the input was the fragments from the golden gate along with the two adapters.
Here are the input parameters from the 4 ligations:
1) 20ul rxn, 1hr incubation w 10min heat inactivation, 400 units ligase
2) 10ul rxn, 1hr incubation w 10min heat inactivation, 400 units ligase
3) 20ul rxn, 2hr incubation w 10min heat inactivation, 400 units ligase
4) 10ul rxn, 2hr incubation w 10min heat inactivation, 400 units ligase
Incubation at room temp for each.
Heat inactivation @ 65˚C for each.
For these ligations, I'm pretty sure I put way too much DNA into the reaction (35.7ng insert, 187ng each adapter). I'm confused about calculating insert:vector ratio because there is no vector in my experiment, I am simply trying to ligate these 3 fragments together. Does anyone have advice (other than adding less DNA)? I am specifically wondering what a good ratio of insert:adapter would be for a ligation with all linear components, and hopefully a linear output. Does the rule of having
For ligation of three or more fragments, there are some discussions that may work better than a regular ligase. It is called Golden Gate Assembly or cloning. The links are, for example (you can also search Golden gate assembly that pulls out many links)
https://www.researchgate.net/post/Why_is_Golden_Gate_so_efficient_for_assembling_many_fragments_but_T4_ligation_isnt
https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly