NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II FS DNA Module (NEB # E7810)

Add the following components directly to the FS Reaction Mixture:

A	B
Component	Volume
FS Reaction Mixture	35 µl
(red) NEBNext Adaptor for Illumina**	2.5 µl
(red) NEBNext Ultra II Ligation Master Mix*	30 µl
(red) NEBNext Ligation Enhancer	1 µl
Total volume	68.5 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 Oligo kit options, which can be found at www.neb.com/oligos

Note

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.

Set a 100 µl or 200 µl pipette to 50 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.

Incubate at 20°C for 0h 15m 0s in a thermocycler with the heated lid off .

Add 3 µl of (red) USER Enzyme to the ligation mixture.

Note

Note: Steps 4 and 5 are only required for use with NEBNext Adaptors. USER enzyme can be found in most NEBNext oligo kits, all options can be found on the Note: Steps 4 and 5 are only required for use with NEBNext Adaptors. USER enzyme can be found in most NEBNext oligo kits, all options can be found on the www.neb.com/oligos page. If you are using the indexed UMI adaptor, USER is not needed. Please see corresponding manual for use with UMI on the E7395 product page under protocols, manuals, and usage tab. page. If you are using the indexed UMI adaptor, USER is not needed. Please see corresponding manual for use with UMI on the E7395 product page under protocols, manuals, and usage tab.

Mix well and incubate at 37°C for 0h 15m 0s with the heated lid set to ≥ 47°C.

DNA is now ready for size selection or cleanup.