POLYMERASE CHAIN REACTION PROTOCOL

©David B. Fankhauser, Ph.D.
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 Reagents for the PCR Reaction
modified from the protocol of Sonia Wallman

This page has been accessed Counter times since 14 Feb. 2003. 
19 January 2001, 13 Feb 01, 3 Jan 03, 19 Feb 03
 Insertion of reaction tubes
into thermocycler

Wear latex gloves at all times when handling DNA.
 

Program the thermocycler with the prescribed step file (see conditions required for given probe set). For D1S80 primers (primers which work well for us), the step file will be:
SEGMENT TEMP. C TIME
min.		 PURPOSE
1 95 5 initial denaturation
2 95 1 cyclic denaturation
3 65 1 annealing of primers
4 72 1 synthesis of DNA
Repeat the 2, 3 and 4 steps for 30 cycles. At the end of the 30 cycles:
TIME DELAY FILE 72 C 10 minutes
Label a 0.5ml PCR tube on the cap of with your initials.
Prepare reaction mix which contains the following per student sample to be run, assuming DNA sample of 5 L (adjust the dH2O for other volumes of DNA):
    17.35 uL sterile dH2O
    3.0 uL 10x PCR buffer
    1.5 uL 50 mM MgCl2
    1.5 uL 10 mM dNTP (contains all four nucleotide triphosphates)
    1.5 uL primer set
Deliver 25 L of reaction mix to each tube on ice.
Add 5 L of your buccal cell DNA from the previous preparation.
Close top of tube securely and mix reagents by vortexing.
Store sample on crushed ice until all samples have been prepared.

 
 
Warm up the thermocycler
Load thermocycler with 0.5ml PCR reaction tubes. 
For hot start, run at 95 C for five minutes, then add 0.15 L Taq polymerase, 5 units/ uL
Run the thermocycler for prescribed number of cycles so that the polymerase chain reaction amplifies the probed segment of DNA. (This usually requires coming back to the lab several hours later)
Add 5 L of 6x loading dye to the PCR sample. Mix by vortexing. Store all samples on crushed ice for minutes or at -20 Centigrade for days until you are ready to cast agarose gel and electrophorese. 
Here are the resulting D1S80 amplified bands from several genetics students

 

*To initiate hot start PCR, denature the DNA by incubating the tubes for 5 minutes at 95° C (segment 1), maintain the tubes at 95° C while you add 0.4 l Taq DNA polymerase to each tube. Do not allow the tubes to cool and do not take time to mix the reaction mixture after adding the Taq polymerase.