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  • Introduction (E5350)

    Introduction

    General Guidelines
    Molar Ratio
    (2 Dimers : 1 Tetramer : 1 DNA Binding Site)
    Because the correct ratio of DNA to protein determines the efficiency of an assembly, the kit components have been formulated to have equal volumes of each added in a reaction. After assembly, the reactions should contain 5–10% unbound DNA to mitigate aggregate formation which occurs when protein is in excess.

    DNA Substrate
    The Nucleosome Control DNA (208 bp) contains one possible binding site for an octamer. This enables visualization by gel shift assay due to the binding of the octamer and a characteristic shift from 208 bp to ~700 bp on a 6% native polyacylamide gel. When using user-supplied substrate DNA, optimization may be required to determine the number of possible binding sites per molecule. A starting point for the amount of user supplied DNA to add per reaction can be determined using the formulas described in the DNA concentration formula section. Then, keeping the amount of DNA constant, the amount of octamer can be varied to find an optimal ratio of DNA to protein. To conserve DNA and protein during optimization, we would recommend the dilution protocol for 25 pmol.

    Other Considerations

    • Because stability can be an issue for the octamer in vitro without DNA, we provide the Dimer and Tetramer separately.
    • Set up each reaction with dH2O, 5 M NaCl, DNA and protein such that the final concentration is 2 M NaCl, noting that the Dimer and Tetramer are supplied in 2 M NaCl containing buffer.
    • Always add the Dimer and Tetramer last!
    • Because nucleosomes can dissociate when too dilute, it is recommended to keep the final protein concentration above 10 µg/ml (1).
    Protocols
    Two different protocols are available. The quickest is the dilution assembly protocol with assembly being ready in less than three hours. It is ideal for optimization since smaller volumes of all components may be used. The dialysis protocol does take longer but the concentration differences from starting material are minimized and it requires less sample handling.

    References
    1. Luger et al. (2004) Methods in Enzymology, 375, 23-62.