Analysis of ligation products by agarose gel electrophoresis

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Analysis of ligation products by agarose gel electrophoresis

Ligation efficiency can be assessed by agarose gel electrophoresis of ligation reaction products. For sample loading, usage of SDS-supplemented loading dye, e.g., 6X DNA Loading Dye & SDS Solution (#R1151) is recommended to eliminate band shift due to T4 DNA ligase binding to DNA.

    Prepare the loading mixture:
    Ligation reaction product         10 µl
    6X DNA Loading Dye & SDS Solution (#R1151)（可以自己配）         2 µl
    Heat the sample for 10 min at 65°C and load.

Analysis of ligation reaction products on an agarose gel

ligation reaction         400 ng of vector and insert in total were used. Real ligation experiments normally use less DNA, therefore bands on a gel may appear at lower intensity.
M – GeneRuler™ DNA Ladder Mix (#SM0331).
1 – Mixture of DNA insert and vector in T4 DNA Ligase Buffer.
2 – Mixture of DNA insert and vector after the ligation sample loaded with 6X DNA Loading Dye (#R0611).
3 – Mixture of DNA insert and vector after the ligation sample loaded with 6X DNA Loading Dye & SDS Solution (#R1141).

Interpretation of results


    Appearance of higher molecular weight bands and decreased intensity of the vector and insert bands indicate successful ligation.
    Unchanged band pattern after ligation indicates unsuccessful ligation.