Contamination is always a factor to take into consideration. If repeater pipettes are used, it is best to prepare the master mix and aliquot it first, then add the template. It is always a good idea to add a negative control, containing just the master mix and run it with the samples as a check for contamination throughout each step of the process. And, of course, wear gloves and work in as sterile an environment as possible.

Scaling of the reaction mix saves time and pipetting larger volumes increases accuracy. Distributing a master mix will result in a loss of volume, so that additional master mix is needed for a fixed number of reactions.

The following is an example of scaling the mix for 48 samples using a DNA concentration of 2 ng/µl. The amount of DNA required for each reaction is 10 ng or 5 µl of volume. For this example, allow for 52 samples which is a 7.7% overage. The total volume of master mix will be 780 µl. The total volume of reaction mix will be 1040 µl, allowing for the later addition of template.

• Thaw reagents and keep on ice.
• Combine reagents in order as described in Table 1.

• Aliquot 15 µl of the master mix into each well.
• Add 5 µl of sterile distilled water to the negative control.
• Add 5 µl of template to each of the sample wells.
• Centrifuge to bring contents to bottom of wells.
• Seal reactions, load in thermal cycler, then amplify using appropriate program.
• Run 2 µl of PCR product on a gel under appropriate conditions to separate fragments.
• Visualize to determine success of PCR.
• Proceed to PCR cleanup.