Ligation inefficiencies with NEB

[Sabre_seq]

Hello All,

I am currently working on a protocol where I will be using NEBNext® Ultra™ II FS DNA Library Prep Kit (Cat # E7805) ligation mix and enhancer as a 2x master mix for adapter ligation.

Now I am including 20x the amount of adapter into the reaction but I am only getting about 30% ligation of my sample. I am looking for a target that is only present in 1/1000 of the time.

Does anyone have any recommendation for using the Ligation mix that NEB provides with this kit?

[nucacidhunter]

I wonder if you are using the whole kit or just using ligation reagents. I would not expect good ligation using only ligation reagents as this kit uses one pot workflow and skipping frag-end repair step may not provide optimum buffer composition for ligation.

[Adamantli]

depends on the input DNA, 20X may not be optimal. I have used this kit for low input DNA of 10 ng, and I found 100-200X gave the best conversion rate.

Hello All,

I am currently working on a protocol where I will be using NEBNext® Ultra™ II FS DNA Library Prep Kit (Cat # E7805) ligation mix and enhancer as a 2x master mix for adapter ligation.

Now I am including 20x the amount of adapter into the reaction but I am only getting about 30% ligation of my sample. I am looking for a target that is only present in 1/1000 of the time.

Does anyone have any recommendation for using the Ligation mix that NEB provides with this kit?

[drdna]

How are you measuring ligation?