Experimental Considerations - NEBNext Library Quant Kit (E7630)
- qPCR is a sensitive DNA detection method. Proper sterile technique and careful pipetting should be used to avoid DNA contamination and ensure accurate quantitation results.
- This kit is used for quantitation of libraries for Illumina sequencing platforms and includes primers that are complementary to the P5 and P7 sequences. Prior to using this kit, confirm that the library samples being tested include these Illumina adaptor sequences.
- Ensure that all kit components are thawed and mixed prior to use.
- Upon first use, when the NEBNext Library Quant Primer Mix is added to the NEBNext Library Quant Master Mix the date should be recorded on the NEBNext Library Quant Master Mix container. Use this notation to verify that the NEBNext Library Quant Primer Mix was added prior to using the mix in subsequent experiments. Once mixed the combined NEBNext Library Quant Master Mix and NEBNext Library Quant Primer Mix is stable for six weeks at 4°C (mix can be stored without freezing for convenience) or seven months at –20°C.
- For each qPCR reaction add 4 μl of NEBNext Library Quant DNA Standards or diluted library to 16 μl of pre-mixed NEBNext Library Quant Master Mix (with primers).
- We recommend inclusion of a no template control (NTC) reaction in addition the NEBNext Library Quant DNA Standards 1–4. The Cq from the NTC will not be used in quantitation analysis, but serves as a valuable control reaction to ensure performance of the kit and absence of sample contamination.
- Accurate library quantitation relies on careful dilution of library DNA samples. Use a 1X concentration of provided NEBNext Library Quant Dilution Buffer and prepare 1:1,000–1:100,000 dilutions carefully.
- We recommend running triplicate reactions for each standard and library sample. This ensures the most accurate quantitation and enables exclusion of outlier traces due to bubble, plate sealing or other problems.
- Multichannel pipettes can be used with the kit, but care should be taken to ensure consistency of volume when loading both the NEBNext Library Quant Master Mix (with primers) and samples into the qPCR plate. For most accurate and consistent results we recommend single channel pipettes.
- When pipetting into the qPCR plate, it is advisable to avoid the formation of bubbles. Centrifugation of sealed qPCR plates for 5 minutes at 2,500–3,000 rpm is recommended to collect samples at the bottom of wells, but is not required. If 1–2 small bubbles are present at the top of the liquid after loading, the assay can proceed as the bubbles will be removed as the plate is heated for the denaturation step of the PCR cycle.
- If quantifying fewer libraries than require a full plate to be loaded, we recommend avoiding the wells along the edges of the plate, as these can be prone to plate-sealing failures during qPCR runs, resulting in outlier traces or evaporated reagents. When run in triplicate, the failed well does not affect results if excluded from analysis.
- Consult the qPCR instrument manual to verify the appropriate ROX solution to be used. A quick recommendation can be found in Table 1 below, however the appropriate ROX amount should be verified for instruments not listed.
- Use the “SYBR Green” or “SYBR/FAM” channel of the qPCR instrument. Only the single channel plate read is necessary for the NEBNext Library Quant Kit, and selecting a single read often results in faster experiment times.
- Denaturation or melt curves may be included in the qPCR cycling protocol, although this will add time to the workflow and likely not provide helpful information about library quality. Extended discussion of melt curves with examples can be found in Chapter 6.1 in the manual.
|Bio-Rad® iQ™ 5, CFX96, CFX384, Opticon
|Applied Biosystems 7500, QuantStudio®, ViiA7™
|Applied Biosystems 7000, 7300, 7700, 7900HT,