MagicTubes for Perfect PCR
"Hot start" effect with ordinary Taq-polymerase
MagicTubes are intended for the performing of the polymerase chain reaction (PCR).
The use of MagicTubes allows:
a) to obtain the "hot start" effect using the ordinary (i.e. unmodified by antibodies or chemically) DNA-polymerases;
b) to reduce nonspecific amplification product caused by low-temperature priming;
c) to increase the reaction specificity;
d) to increase the yield of the target PCR product.
The advantages of the MagicTubes can be especially revealed at the PCR amplification of "difficult" templates (GC-rich DNA sequences, templates with complex structure, etc.), when performing the multiplex PCR and at the amplification of small quantities of the initial DNA.
The MagicTubes are easy to use. They can be described as the ordinary PCR-tubes, inner walls of which are coated with a special polymer. Acting together with MagicBuffer it optimises the PCR conditions. The optimal efficiency of the MagicTubes is reached by the 5-minutes preheating of the full PCR mixture at the temperatures of 92-95°C during the first cycle of the PCR.
In order to achieve the best results the PCR should be performed with the 10x-reaction Magic-buffer provided with the MagicTubes. The above-mentioned reaction buffer was specially developed for the performing of the PCR with Taq-polymerase in the MagicTubes.
The performing of the "hot start" in the MagicTubes instead of wax-barrier, manual, or antibody-mediated "hot starts" to prevent nonspecific amplification and primer-dimer formation, provides enhancing the specificity and sensitivity of PCR. Since no extra reagents (as antibody) is added during setup, there is no risk of contamination of eucariotic DNA traces from antibody solution or due to re-opening reaction tubes after heating as in manual "hot start", and there is no extended high-temperature reactivation as in "hot start" performing by chemically modified DNA polymerases.
1. The 10X reaction MagicBuffer provided is optimized for the application with Taq polymerase.
2. The reaction "hot start" is achieved by the 5-minutes preheating at the temperature of 92-95°C during the first PCR cycle.
3. When the 10X MagicBuffer is used, Mg++ should not be added to the reaction mixture.
4. The recommended volume of the reaction mixture is 20-80 microliters.
Increased specificity of PCR in MagicTubes
A 180-bp DNA fragment was amplified from 50 ng of human genomic DNA for 30 cycles. “Hot start” was performed manually (the enzyme was brought in into the 94°C preheated reaction mixture) (lanes 1, 2) or with the use of MagicTubes (lanes 3, 4). The PCR was performed in the volume of 25 µl containing 0.5 activity units (lanes 1, 3) or 2.0 activity units (lanes 2, 4) of Taq polymerase accordingly.
Lane 5: molecular weight markers.