1. In a sterile, nuclease-free microcentrifuge tube, combine the following on ice:
1) by 5×Direct-Loading PCR Buffer reaction
2) by 10×PCR Buffer with Mg2 reaction
3) by 10×PCR Buffer without Mg2 reaction
Note: thaw completely and vortex thoroughly prior to use.
2. Perform PCR using your standard parameters. An example profile is given in above tables. For the cycling protocol, we recommend the following:
Note: if using a thermal cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation during thermal cycling. Centrifuge the reactions in a microcentrifuge for 5 seconds.
3. Separate the PCR products by agarose gel electrophoresis and visualize with ethidium bromide or any other means. For reactions containing the 5×Direct-Loading PCR Buffer, load the reaction onto the gel directly after amplification. Reactions containing the 10×PCR Buffer with Mg2 or 10×PCR Buffer without Mg2 will need to add 6×loading buffer to electrophoresis.
The above cycling conditions were established and tested using a MJ Rearch PTC-200 .You may need to adjust these conditions for other thermal cyclers.