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  • RNase Contamination Assay Kit (E3320)

    Protocol

    1. Preparation of test samples

      Each 10 µl standard reaction uses 1 µl of test sample. The test sample should be diluted in sample storage buffer if it is very concentrated (e.g. enzyme stock). It is recommended that a reaction with a sample diluent (e.g. enzyme storage buffer) as well as a water control should be included along with test samples. The recommended minimal reactions are three.

      If a custom reaction buffer is used for sample testing, replace 10X NEBuffer 4 with 10X custom reaction buffer. Be sure to include appropriate controls. If the test sample is water, we recommend replacing the water volume with sample water (8 µl). Be sure to include a control reaction with RNase-free water. Samples containing very high concentration of salt or organics may inhibit RNase activity and interfere with PAGE.

    2. Assay set up

      It is highly recommended that a master mix with enough overage is prepared. Table 1 shows an example of a master mix preparation. After combining the components, mix them thoroughly by vortexing or by flicking the tube several times and then pulse-spin briefly. Aliquot 9 µl of master mix to each tube and then add 1 µl of appropriate controls and test samples to the respective tubes. Mix well.

      Table 1. Reaction Set Up for RNase Contamination Assay
      Componenets Each Rxn 10 Reactions with
      10% Overage
      Water 7μl 77μl
      10X NEBuffer4 1 μl 11 μl
      RNA probe 1 μl 11 μl
      Total volume 9 μl 99μl
    3. Incubate the reactions in a dry air incubator at 37°C for 1 – 16 hours (overnight)

      Incubation time varies depending on the quality requirements of test samples. More specifically, it depends on the applications of the test sample. For example, if the NTPs will be used in in vitro transcription at 37°C for 16 hours, the NTPs should be incubated for 16 hours. Because the assay is more sensitive when detergent is present, 2 hour incubation is sufficient for samples containing detergent.

    4. Proteinase K treatment to release bound RNA probe (optional)

      If the test sample can potentially bind to the RNA probe, e.g. MMLV reverse transcriptase, the test reaction should be treated with 1 µl of proteinase K at room temperature for 5–10 minutes before gel analysis. This treatment will release the bound RNA probe and help with gel image analysis. You may choose to treat all of the reactions with proteinase K when you are unsure whether the test samples will bind to the RNA probe. Proteinase K treatment does not interfere with the assay. Figure 1A (see product page) shows the advantage of PK treatment of reactions with samples that bind to the RNA probe (reactions 1 and 2) and no difference for reactions with samples that do not bind to RNA probe (control and reaction 3).

    5. Stop reaction by adding 10 µl of 2X RNA loading dye to each reaction

      Add 10 µl of 2X RNA loading dye to each reaction. Mix thoroughly and pulse-spin briefly. If you are unable to proceed with gel analysis, store the samples at -20°C. Samples can be stored at -20°C for at least three days. 

    6. Gel electrophoresis

      We recommend analyzing the reactions on a denaturing polyacrylamide gel (PAGE-Urea gel, 5–10%). Before loading, heat the samples at 65–70°C for 5–10 min, pulse-spin briefly. Load 10 µl of each sample onto gel. Run the gel until the bromophenol blue (the fast dye) is approximately 2–3 cm from the bottom of the gel. If the gel runs too far, the free label will run off the gel. For a standard mini-gel, the running time is about 30–45 min. 

    7. Visualization and analysis

      a) On a fluorescein capable imaging system
      The integrity of the fluorescein labeled RNA probe can be visualized by a fluorescein capable imaging system directly. After electrophoresis, remove the gel carefully and scan it on the Typhoon scanner or equivalent. Compare the RNA probe intensities of test samples to that of a valid water control. If the RNA probe is mostly intact, the sample passes the RNase contamination assay (e.g. reactions 2 and 3 in Figure 1A (see product page)). 

      b) By SYBR Gold staining
      If a Typhoon scanner is not available the gel can be stained with SYBR Gold and visualized on a UV light box. Carefully remove the gel and stain it in 1X SYBR Gold for 5–10 min with occasional shaking. Rinse the gel briefly with water and visualize it on a UV light box. Figure 1B (see product page) shows the image of the same gel in Figure 1A (see product page) stained by SYBR Gold. Note in Figure 1B, the free label in reaction 1 is barely visible. Since SYBR Gold stains nucleic acids, samples containing DNA or RNA will interfere with the visualization.