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.


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.

  1. 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
    Total volume   83.5 μl
    *The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500) Oligos for Illumina.
  2. Mix by pipetting, followed by a quick spin to collect all liquid from the sides of the tube.
  3. Incubate at 20°C for 15 minutes in a thermocycler.
  4. Add 3 μl of USER™ enzyme to the ligation mixture from step 3.
  5. Mix well and incubate at 37°C for 15 minutes.
  6. DNA is now ready for size selection or clean-up using Agencourt® AMPure XP beads or a Qiagen® PCR column.