Prevention of unexpectedly long PCR products primed at short inverted repeats.

ers of <20 μL often leak while 30 μL of wax results in approximately a 2 mm space between the upper and lower aqueous reagents. Second, the temperature at which mixing occurs depends on both the melting point of the wax and the amount of wax present. We have found that the upper aqueous layer does not enter the bottom layer until the tube has reached >80°C. We determined when the upper aqueous layer mixed with the bottom aqueous layer by withdrawing tubes from the PCR machine at various temperatures and visually determining when the bright red upper aqueous phase entered and mixed with the bottom aqueous layer. Thus, while higher melting point paraffin waxes are available, their use is not necessary to hot-start RT-PCR at temperatures above a reasonable annealing temperature (generally 55°– 65°C). In addition, higher melting point waxes are more difficult to apply to tubes and are much harder to puncture than the 54°–61°C paraffin wax used in this study. We found that the method described here reduces false priming (primer dimers and laddering) with several primer sets used by our group.