e following steps 13
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
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
Source:Page: All 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Related biology technology :1
. HotStarTaq DNA Polymerase2
. QIAGEN Multiplex PCR Handbook3
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. Transfection Reagent Selector Kit Handbook