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  • FAQ: How to prepare denatured RNA samples to be run on a native gel?

    Two methods are recommended for sample preparation:

    Method 1:
    This method utilizes the 2X ssRNA Ladder Sample Buffer provided, and samples should be run on a native gel prepared with 1X TBE. This method does not always denature RNA molecules completely.

    1. Combine on ice:
    Low Range
    ssRNA Ladder (500 µg/ml): 2 µl (1 µg)
    H2O (RNase-free): 3 µl
    2X RNA Ladder Sample Buffer: 5 µl
    Total Volume 10 µl
    2. Heat at 65°C for 5 minutes, chill on ice, load entire sample on gel.

    Method 2:
    This method utilizes formamide and formaldehyde in the sample as denaturants, and samples should be run on a native gel prepared with 1X MOPS buffer. This method is the most effective for denaturing RNA and should be used for precise sizing. The more denatured the RNA, the lower its affinity for EtBr, therefore this method requires 2-3 µg of ladder to be loaded to give good band intensity.

    1. Combine on ice:
    Low Range
    ssRNA Ladder (500 µg/ml): 4-6 µl (2-3 µg)
    H2O: up to 6 µl
    10X MOPS: 2 µl
    Deionized Formamide: 10 µl
    Formaldehyde (37%)
    2. Heat at 70°C for 5 minutes, chill on ice. Add 2 µl of 6X loading dye with bromophenol blue, load entire sample.