DNA Engine Tetrad 2 Thermal Cycler Operations Manual
4-8
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Sealing Sample Vessels
To avoid changing the concentration of reactants, steps must be taken to prevent the
evaporation of water from reaction mixtures during thermal cycling. Only a layer of oil or
wax will completely prevent evaporation from the surface of the reaction fluid. However, an
adequate degree of protection can be achieved by sealing vessels with caps, film, adhesive
seals, or mats, then cycling the samples using the heated lid to prevent condensation.
Sealing with Oil or Wax
Mineral oil, silicone oil, paraffin wax, or Chill-out
™
liquid wax may be used to seal sam-
ples. Use only a small amount of oil or wax; 1–3 drops (15–50 µl) are usually sufficient.
(Include this volume in the total volume when setting up a calculated-control protocol;
see “Choosing a Temperature Control Mode” in Chapter 5.)
Use the same amount of
oil or wax in all sample vessels to ensure a uniform thermal profile.
Most paraffin waxes solidify at room temperature. The wax can then be pierced with a
micropipette tip and the samples drawn off from below the wax. Silicone oil and mineral
oil can be poured off or aspirated from tubes if the samples are first frozen (–15° to –20°C).
The samples are usually pure enough for analysis without an extraction.
Chill-out liquid wax (available from Bio-Rad) is an easy-to-use alternative to oil. This puri-
fied paraffinic oil solidifies at 14°C and is liquid at room temperature. By programming a
hold at low temperature, the wax can be solidified at the end of a run. A pipette tip can
then be used to pierce the wax in the tubes and remove the samples. The wax is available
in a clear, optical-assay grade or dyed red to assist in monitoring its use. The red dye has
no adverse effects on fluorescent gel analysis of reaction products.
Sealing with the Hot Bonnet Lid
The Hot Bonnet’s heated inner lid maintains the air in the upper part of sample ves-
sels at a higher temperature than the reaction mixture. This prevents condensation of
evaporated water vapor onto the vessel walls and lid, so that solution concentrations
are unchanged by thermal cycling. The Hot Bonnet lid also exerts pressure on the
tops of vessels loaded into the block, helping to maintain a vapor-tight seal and to
firmly seat tubes or the plate in the block.
Caps, film, adhesive seals, or mats must be used along with the Hot Bonnet lid to
prevent evaporative losses.
Note:
When tubes are cooled to below-ambient temperatures, a ring of condensa-
tion may form in tubes above the liquid level but below the top of the sample block.
This is not a cause for concern since it occurs only at the final cool-down step, when
thermal cycling is complete.
Microseal
®
'A' film offers a quick alternative to sealing microplates or arrays of tube strips.
This film is specially designed to seal tightly during cycling, yet release smoothly to
minimize the risk of aerosol formation and cross-contamination of samples.
Microseal 'A' film is easily cut for use with fewer than 96 samples.
