Molecular Techniques and Methods

PCR Radioactive Labeling
to Generate A High Specific Activity Probe

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Using the protocol provided, typically greater than 80% of the labeled deoxyribonucleotide is incorporated, generating probes of highest specific activity (routinely 109cpm/ug).




MATERIALS AND SOLUTIONS

PCR Mix (300 ul)
10 x Taq DNA Polymerase Buffer ----------------------- 50 ul
25 mM MgCl2 ------------------------------------------- 50 ul
20 uM dNTPs w/o dCTP -------------------------------- 50 ul
2 uM PCR Primer1 -------------------------------------- 50 ul
2 uM PCR Primer2 -------------------------------------- 50 ul
Deionized H2O ------------------------------------------ 50 ul
  • Store at -20oC for 1 month.




    PROCEDURES

    1. Add the followings in a 200 ul PCR tube.

    PCR Mix
    11.5 ul
    plasmid DNA or gel purified insert DNA (1-10 ng/ul)
    1 ul
    Taq DNA Polymerase (5 U/ul)
    0.5 ul
    Fresh [a-32P]dCTP (3,000-6,000 Ci/mmole and 10 mCi/ ml)
    5 ul

  • 800 Ci/mmole dCTP is not good enough to generate high specific activity probes.


    2. Overlay 50 ul mineral oil to prevent radioactive contamination.

    3. Do PCR reaction as follow.

    1 cycle
    Denaturation
    95oC
    2 min
    35 cycles
    Denaturation
    95oC
    10 sec
    Annealing
    Tm-5oC
    20 sec
    Extension
    72oC
    1 min/kb
    1 cycle
    Final Extension
    72oC
    7 min

  • Extension time depends on DNA length. Generally, 1 minute per 1 kb DNA.


    4. Remove unincorporated [a-32P]dCTP.

    5. Optional;
    Check the incorporation of radioactivity by TCA precipitation.

    - Spot 1 ul of PCR reaction on Whatman No.1 filter.
    - Agitate filter in ice cold 10% TCA (containing 1% Na-pyrophosphate) for 5 minutes.
    - Wash filter in 5% TCA for 5 minutes. Repeat 3 times.
    - Wash filter in 100% ethanol for 5 minutes.
    - Air dry filter and count incorporation in liquid scintilation counter.





    NOTES

  • For the best result, DNA to be labeled should be digested out from vector plasmid DNA and gel-purified.

  • To get the highest specific activity, [a-32P]dCTP should be less than 1 week old.

  • It is good practice to overlay the mineral oil in PCR tubes to prevent the radioactive contamination in wells of PCR machine.

  • Tm of primer can be calculated as following.
    Tm (oC) = [(G+C) x 4] + [(A+T) x 2]


  • KIT INFORMATION



    REFERENCES


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

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