( e following steps 13 ...)HotStarTaq PCR Handbook

e following steps 13 in protocols.
Add 25 l from the RT reaction to each PCR tube containing the master mix.
Continue with step 5 in the PCR protocols. Oligotex is not available in Japan.
* For further information about RNeasy, Oligotex, and Omniscript products, contact your local QIAGEN Technical Services or distributor (see inside front cover).
Please refer to the manufacturers instructions for the amount of enzyme required.

6. Touchdown PCR

Touchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of PCR. The annealing temperature in the initial cycle should be 510C above the Tm of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 12C/cycle until a temperature is reached that is equal to, or 25C below, the Tm of the primers. Touchdown PCR enhances the specificity of the initial primertemplate duplex formation and hence the specificity of the final PCR product.

To program your thermal cycler for touchdown PCR, you should refer to the manufacturers instructions.

7. Purification of PCR products

After amplification, the PCR sample contains a complex mixture of specific PCR product and residual reaction components such as primers, unincorporated nucleotides, enzyme(s), salts, mineral oil, and probably nonspecific amplification products. Before the specific PCR p
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