From 2007.igem.org

Protocol for PCR with Taq DNA Polymerase Avoiding Contamination PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.

DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing the reaction mixture. Fresh gloves should be worn for DNA purification and each reaction set-up. The use of dedicated vessels and positive displacement pipettes or tips with aerosol filters for both DNA sample and reaction mixture preparation, is strongly recommended. The reagents for PCR should be prepared separately and used solely for this purpose. Autoclaving of all solutions, except dNTPs, primers and Taq DNA Polymerase is recommended. Solutions should be aliquoted in small portions and stored in designated PCR areas. Aliquots should be stored separately from other DNA samples. A control reaction, omiting template DNA, should always be performed, to confirm the absence of contamination. These are only rough guidelines. Detailed instructions about PCR laboratory setup and maintenance may be found in PCR Methods and Applications, 3, 2, S1-S14, 1993.

Preparation of Reaction Mixture To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs, primers and Taq DNA Polymerase in a single tube, which can then be aliquoted into individual tubes. MgCl2 and template DNA solutions are then added. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers.

Reaction Mixture Set Up

Gently vortex and briefly centrifuge all solutions after thawing.

Add, in a thin-walled PCR tube, on ice:

Reagent ;	Final concentration	Quantity, for 50 µlof reaction mixture
Sterile deionized water	-	variable
10X Taq buffer	1X	5 µl
2 mM dNTP mix	0.1-1 µM	variable
Primer I	0.1-1 µM	variable
Primer II	1.25 u / 50 µl	variable
Taq DNA Polymerase	1.25 u / 50 µl	variable
25 mM MgCl2	1-4 mM	variable*
Template DNA	10pg-1 µg	variable

 * Table for selection of 25 mM MgCl2 solution volume

Final concentration of MgCl2 in 50 µl reaction mix, mM	1.0	1.25	1.5	1.75	2.0	2.5	3.0	4.0
Volume of 25 mM MgCl2, µl	2	2.5	3	3.5	4	5	6	8