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Old 01-03-2019, 07:48 AM   #1
Junior Member
Location: USA

Join Date: Nov 2018
Posts: 1
Post Ligation inefficiencies with NEB

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?
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Old 01-03-2019, 04:11 PM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,199

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.
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