Protocol for use with NEBNext Ultra II End Repair/dA-Tailing Module (E7546) and NEBNext Ultra II Ligation Module (E7595)

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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.
Colored bullets indicate the cap color of the reagent to be added to a reaction.
Stopping points in the protocol.


Starting Material:

500 pg–1 µg fragmented DNA that has been end repaired and dA-Tailed using the NEBNext End Repair/dA-Tailing Module (NEB #E7546).

 If DNA input is ≤ 100 ng, dilute the NEBNext Adaptor for Illumina in 10 mM Tris-HCl or 10 mM Tris-HCl with 10 mM NaCl as indicated in Table 1.1.

1.1. Add the following components directly to the End Prep Reaction Mixture:

Table 1.1: Adaptor Dilution 

INPUT   ADAPTOR DILUTION (VOLUME OF ADAPTOR: TOTAL VOLUME)  WORKING ADAPTOR CONCENTRATION  
1 µg–101 ng No Dilution
15 µM
100 ng–5 ng
10-Fold (1:10)
1.5 µM
less than 5 ng  25-Fold (1:25)
0.6 µM   

End Prep Reaction Mixture
60 µl 
(red) NEBNext Ultra II Ligation Master Mix*  30 µl 
 (red) NEBNext Ligation Enhancer  1 µl 
 (red) NEBNext Adaptor for Illumina** 
2.5 µl 
Total volume
93.5 µl

* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535, and #E6609) Oligos for Illumina.

Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.

1.2. Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

(Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).

1.3. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.

1.4. Add 3 µl of  (red) USER™ Enzyme to the ligation mixture from Step 1.3.

Note: Steps 1.4 and 1.5 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600 and #E6609) Oligos for Illumina.

1.5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.

1.6. DNA is now ready for size selection or cleanup.

Note: Please see NEB #E7645 manual for recommended size selection/cleanup and PCR amplification protocols.

 Samples can be stored overnight at –20°C.