II: DNA Amplification by PCR
- Use a
micropipet with a fresh tip to add 22.5 µl 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.
- Use fresh
tip to add 2.5 µl 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.
the cap of your tube with a number, as assigned by your teacher. In
this way, your results will be anonymous.
- 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.
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 4°C to hold samples
after completing the cycle profile, but amplified DNAs also hold well
at room temperature.
Denaturing time and temperature 30 sec - 94°C
Annealing time and temperature 30 sec - 58°C
Extending time and temperature 30 sec - 72°C
Hold at -4°C (optional)