Molecular Techniques and Methods

Biotin- or, Digoxigenin-dUTP Labeling of DNA by PCR

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This labeling reaction produces 50 ul of Biotin- or, DIG-labeled probe solution, which is enough to perform 25 in situ hybridization.



MATERIALS AND SOLUTIONS

10 x PCR Buffer (without MgCl2) (1 ml)
100 mM Tris-HCl (pH 8.3) -------------------------------- 100 ul of 1 M Tris-HCl
500 mM KCl ------------------------------------------------ 500 ul of 1 M KCl
Sterile H2O -------------------------------------------------- 400 ul


10 x PCR Biotin- or, DIG-Labeling Mix (70 ul)
2 mM dATP ------------------------------------------------ 1.4 ul of 100 mM dATP
2 mM dCTP ------------------------------------------------ 1.4 ul of 100 mM dCTP
2 mM dGTP ------------------------------------------------ 1.4 ul of 100 mM dGTP
1.3 mM dTTP ---------------------------------------------- 1 ul of 100 mM dTTP
0.7 mM Biotin-, or DIG-dUTP -------------------------- 50 ul of 1 mM Biotin- or, DIG-11-dUTP
10 mM Tris-HCl (pH 7.0) --------------------------------- 14.8 ul




PROCEDURES

1. Add the following components to PCR tube.

10 PCR Buffer
5 ul
25 mM MgCl2
4 ul
10x PCR Biotin- or, Dig-Labeling Mix
5 ul
2 uM Forward primer
5 ul
2 uM Reverse Primer
5 ul
Plasmid DNA
10-20 pg
Taq polymerase
0.5 ul
H2O
Make final vol. to 50 ul


2. Do the PCR reaction. Use the following thermal profile.


1 cycle
Denaturation
95oC
2 min
40 cycles
Denaturation
95oC
15 sec
Annealing
Tm-5oC
15 sec
Extension
72oC
1 min/ kb
1 cycle
Final Extension
72oC
10 min


  • Tm of PCR Primer = [2 x (A+T)] + [4 x (C+G)]


    3. Remove unincorporated dNTPs by Sephadex G-50 column.

    Alternatively,
    4. To the labeled DNA, add 5 ul 4 M LiCl and 150 ul prechilled (-20oC) 100% ethanol.

    5. Let the precipitate form for at least 30 min at -70oC or 2 h at -20oC.

    6. Centrifuge the tube (at 13,000g) for 30 min at 4oC.

    7. Discard the supernatant.

    8. Wash the pellet with 100 ul ice-cold 70% (v/v) ethanol.

    9. Centrifuge the tube (at 13,000g) for 5 min at 4oC.

    10. Discard the supernatant.

    11. Dry the pellet under vacuum.

    12. Dissolve the pellet in 50 ul TE (pH 8.0) buffer.

    13. Dilute an aliquot of the probe solution in the hybridization buffer to be used for the in situ hybridization.
    (If you are not going to use the probe immediately, store the probe solution at -20oC.)




    NOTES

  • To produce less probe, scale the reaction volume and components down proportionally.


  • The concentration of MgCl2 must be determined empirically.
    Initially, try 1.5 mM MgCl2. MgCl2 can be added up to 6 mM in final concentration.


  • The concentration of PCR primers must be determined empirically.
    Try a 0.2-0.5 mM concentration of each primer in the reaction mixture.


  • The amount of template DNA must be determined empirically.
    Initially, try 50 ng eukaryote genomic DNA or 50 pg plasmid DNA.


  • The amount of Taq DNA polymerase must be determined empirically. Initially, try 2.0 units Taq DNA Polymerase.


  • Tm (oC) = [Sum(G/C) x 4] + [Sum(A/T) x 2]


  • Optional - Overlay with 100 ul mineral oil to reduce evaporation of the mixture during amplification.


  • Drying the pellet is important because small traces of residual ethanol will cause precipitation if the hybridization mixture contains dextran sulfate. Trace ethanol can also lead to serious background problems.


  • If the hybridization buffer contains a high percentage formamide, dissolve the probe pellet in a smaller amount of TE buffer to form a more concentrated probe stock solution.


  • Avoid repeated freezing and thawing of the probe.


  • The amount of probe solution to use in the hybridization reaction must be determined empirically. Initially, try using 2 ul of probe solution (out of the original 50 ul total) in 20 ul hybridization solution for each in situ hybridization (under a 24 X 24 mm coverslip). If the probe was dissolved in 50 ul TE, add correspondingly less of the concentrated probe stock to the hybridization buffer.


  • The DIG-dUTP used in this labeling reaction is alkali-labile. Avoid exposing the probe to strong alkali (e.g., 0.2 mM NaOH). Standard in situ hybridization procedures should not detach the DIG label from the probe.





  • KIT INFORMATION




    REFERENCES

  • Innis, M.A., Gelfand, D.H., Sninski, J.J., and White, T.J., 1990, PCR Protocols: A Guide to Methods and Applications. San Diego, CA, Academic Press.


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

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