Protocol for DNA (E7445)
This caution sign signifies a step in the protocol that has multiple paths
leading to the same end point but is dependent on a user variable, like
the amount of input DNA.Protocol
Starting Material: 5 ng–1 μg of fragmented DNA that has been end repaired and dA-Tailed using the NEBNext End Repair/dA-Tailing Module (NEB #E7442)Adaptor Ligation
If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina* 1:10
in sterile water and use immediately to a final concentration of 1.5 μM.- Add the following components directly to the End Prep reaction mixture
(65 μl) and mix well:
Blunt/TA Ligase Master Mix 15 μl
NEBNext Adaptor for Illumina*2.5 μl
Ligation Enhancer 1 μl
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Total volume 83.5 μl
*The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500) Oligos for Illumina. - Mix by pipetting, followed by a quick spin to collect all liquid from the sides of the tube.
- Incubate at 20°C for 15 minutes in a thermocycler.
- Add 3 μl of USER™ enzyme to the ligation mixture from step 3.
- Mix well and incubate at 37°C for 15 minutes.
- DNA is now ready for size selection or clean-up using Agencourt® AMPure XP beads or a Qiagen® PCR column.
