NEBNext Library Quant Kit Quick Protocol (E7630)

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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.

If this is your first time using this kit, we strongly recommend using the full protocol found in the NEBNext Library Quant Kit Instruction Manual.

Starting Material:
(5 ng–1 μg) of fragmented DNA

1.1 Thaw Kit Reagents

Thaw kit components at room temperature. Mix well and centrifuge briefly
to collect material from the sides of the tubes. Place reagents on ice.

1.2 Prepare Master Mix + Primer Mix

NEB #E7630S : Add 100 μl NEBNext Library Quant Primer Mix to the tube of NEBNext Library Quant Master Mix (1.5 ml). Mix by vortexing for 10 seconds. Write the date on the master mix tube to indicate that primer mix has been added.

NEB #E7630L : Add 500 μl NEBNext Library Quant Primer Mix to the bottle of NEBNext Library Quant Master Mix (7.5 ml). Mix by vortexing for 10 seconds. Write the date on the master mix bottle to indicate that primer mix has been added.

Note: If using ROX for normalization, add to the NEBNext Library Quant Master Mix. Add 20 μl (NEB #E7630S ) or 100 μl (NEB #E7630L ) ROX and vortex. See Table 1 in Chapter 3 for ROX selection.

1.3 Prepare NEBNext Library Quant Dilution Buffer (1X)

Prepare the NEBNext Library Quant Dilution Buffer (1X) by making a 1:10 dilution of the 10X buffer in nuclease-free water. Prepare sufficient buffer for the desired number of libraries to be quantitated, allowing 1.2 ml for each library.

1.4 Prepare Library Dilutions

Prepare an initial 1:1,000 dilution of each library sample in NEBNext Library Quant Dilution Buffer (1X).

  1. Add 1 μl library sample to 999 μl NEBNext Library Quant Dilution Buffer (1X) to create a 1:1,000 dilution.

    Prepare two additional dilutions of the diluted Library samples to create 1:10,000 and 1:100,000 dilutions. These two library dilutions will be used for qPCR analysis.

  2. Add 10 μl of the 1:1,000 dilution to 90 μl NEBNext Library Quant Dilution Buffer (1X) (creates 1:10,000 dilution).

  3. Add 10 μl of the 1:10,000 dilution to 90 μl NEBNext Library Quant Dilution Buffer (1X) (creates 1:100,000 dilution).
1.5 Prepare qPCR Assays

For best results, we recommend running each DNA standard and library sample in triplicate.

  1. Prepare DNA standards and diluted library samples.
    NEBNext Library Quant Master Mix (with primers) 16 μl
    DNA standard or library dilution 4 μl
    --------------------------------------------------------------------------------
    Total volume 20 μl

  2. Prepare a no-template control.
    NEBNext Library Quant Master Mix (with primers) 16 μl
    Library Dilution Buffer (1X) 4 μl
    --------------------------------------------------------------------------------
    Total volume 20 μl

    Mix reactions by pipetting sample or buffer at least 5X. Try to minimize bubbles in plate wells, but 1–2 bubbles per well will be removed by heating and not affect results. If replicates are prepared outside of the qPCR plate, load 19 μl per well to minimize bubble formation.

1.6 Run qPCR Assay in a Real-time Thermal Cycler Using FAM/SYBR Setting

  1. qPCR cycling conditions
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 95°C 1 minute 1
    Denaturation
    Extension
    95°C
    63 °C
    15 seconds
    45 seconds
    35

    Note: A denaturation/melt curve can be included if desired, but is optional. See discussion in Section 6.1.

    We recommend the “Fast” temperature profile where applicable (e.g. Applied Biosystems® StepOne Plus®, 7500 Fast) if appropriate plates are available. “Standard” mode is compatible with the kit if desired.

1.7 Analyze Data and Calculate Library Concentrations

Use the real-time instrument to annotate the standard concentrations as follows:

SAMPLE NAME CONCENTRATION (pM)
DNA Standard 1 10
DNA Standard 2 1
DNA Standard 3 0.1
DNA Standard 4 0.01

Confirm PCR efficiency for the DNA standards is 90–110%.

Adjust library concentration for size using:
Adjusted Conc. = Calculated Conc. × 399 / library size (bp)

Calculate concentrations using standard curve generated with qPCR instrument software, or use NEB qPCR webtool to calculate standard curve and library concentrations, available at http://nebiocalculator.neb.com/qPCR