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L A B O R A T O R Y

Part II: DNA Amplification by PCR

Reagents Equipment & Supplies Shared Items
Primer/loading buffer mix, 22.5 l
Human DNA, 2.5
l
Ready-to-Go Bead (in reaction tube)
Mineral oil (depending on thermal cycler used)

1-20 l micropipet and tips Thermal Cycler

Procedure

  1. Use a micropipet with a fresh tip to add 22.5 l of the appropriate primer/loading buffer mix to a PCR tube containing a Ready-To-Go PCR Bead. Tap tube with finger to dissolve bead.
  2. Use fresh tip to add 2.5 l of human DNA (from Part I) to reaction tube, and tap to mix. Pool reagents by pulsing in a microcentrifuge or by sharply tapping tube bottom on lab bench.
  3. Label the cap of your tube with a number, as assigned by your teacher. In this way, your results will be anonymous.
  4. Add one drop of mineral oil on top of reactants in the PCR tube. Be careful not to touch the dropper tip to the tube or reactants, or subsequent reactions will be contaminated with DNA from your preparation. Note: Thermal cyclers with heated lids do not require use of mineral oil.
  5. Store all samples on ice until ready to amplify according to the following profile. Program thermal cycler for 30 cycles according to the following cycle profiles. Each program may be linked to a 4C to hold samples after completing the cycle profile, but amplified DNAs also hold well at room temperature.

    Denaturing time and temperature 30 sec - 94C
    Annealing time and temperature 30 sec - 58C
    Extending time and temperature 30 sec - 72C
    Hold at -4C (optional)

M I T O C H ON D R I A L  C O N T R O L  R E G I O N

2000, DNA Learning Center, Cold Spring Harbor Laboratory
Noncommercial, educational use only.
Kits available from Carolina Biological Supply Company.