Molecular Techniques and Methods

Removal of Unincorporated dNTPs from
Labeling Reaction Using Home-Made Apparatus

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION





MATERIALS AND SOLUTIONS

Color Indicator Solution (1 ml)
2.5% Blue Dextran
0.5% Bromo Phenol Blue
  • Dissolve in deionized H2O


    Bio-Gel P-60 Suspension (Bio-Rad)
    Bio-Gel P-60 ------------------------------------- 1 g
    Distilled H2O ------------------------------------- 20 ml
  • Allow to swell Bio-Gel P-60 overnight before use.




    PROCEDURES

    1. Place a small amount of glass wool at the bottom of pasteur pipette.

    2. Load the Bio-Gel P-60 into the pasteur pipette.

    3. Fill the Bio-Gel P-60 upto 1/2-2/3 height of pasteur pipette.

    4.To 50 ul of labelling reaction, add 10 ul of Color Indicator Solution.

    5. Mix well using pipettor.

    6. Load the labelling reaction mixture at the top of the Bio-Gel.

    7.Collect the solution containg labeled probe, where
    Blue Dextran (greenish blue) coming out.

    8. Discard pasteur pipette according to the radiation safty regulation.




    NOTES

  • Glass wool is a potential carcinogen. For the safty, work with glass wool in the ventilating hood.


  • In Bio-Gel P-60 column chromatography, large fragments elute first.


  • Blue Dextran behaves as like 300-600 nucleotieds in Bio-Gel P-60 column chromatography.


  • Unincorporated dNTPs stay with bromo phenol blue.



  • KIT INFORMATION


    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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