# Guidelines for Using the Single-use, Indexed 5′ Adaptor Plate (NEB #E9500 and #E9530)

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 Colored bullets indicate the cap color of the reagent to be added.

2.1. Setting up the 5′ Adaptor Ligation with Sample Indexing

2.1.1.  Thaw the NEBNext Direct GS Indexed 5′Adaptor Plate for 10–15 minutes at room temperature.

2.1.2.  Remove the hard plastic plate cover. Briefly centrifuge the plate (280 x g for ~1 min) to collect all of the adaptor at the bottom of each well.

2.1.3.  Orient the  (red) NEBNext Direct GS Indexed 5′ Adaptor Plate as indicated in Figure 2.1 (red stripe towards the user). With a pipette tip, pierce the desired well(s) (Figure 2.1A) and transfer 5 μl of the indexed adaptor to each sample. It is important to change pipette tips before piercing a new well to avoid cross contamination of indexed adaptors. Alternatively, the wells can be pierced using the bottom of clean PCR strip tubes (see Figure 2.1B) prior to pipetting the 5′ adaptor. Use a new, clean strip tube for each new well to be pierced.

Note: There is enough adaptor in each well for one 5′ ligation with Sample Indexing reaction. Do not reuse adaptor if the seal has been previously pierced to avoid contamination with other indexed adaptors.

2.1.4.  Proceed with the 5′ Ligation with Sample Indexing reaction according to Section 1.3.