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PNA Molecular Beacons
The mastermix for the PCR was prepared using Buffer 1X, MgCl 2 2mM, dNTPs 200 µM,
Primer For and Rev 400 nM and Taq 0.05U/µl. The primer uses were:
Forward 5’-TCTGTGGAGAAGAGCTACGAGTTG-3’, Reverse: 5’-
AGGCTGGTAAAGAACCTCAGGACA-3’
The PCR products were immediately analyzed by 2% agarose gel in 0.5× TBE or stored at
−20°C until use. In the unbalanced PCR a first amplification was performed as described
above, then a small amount (2–5 µL) of the reaction mixture was amplified in the second run
using a fresh mastermix solution of the same composition except that a 10 fold excess of the
primer on the target sequence was used.
In order to prepare the samples for the HPLC analysis, two tubes containing 50 µl of PCR
product each, were purified by Genuine PCR Clean-up Kit from Sigma-Aldrich and
concentrated redissolved in 50 µl of water. 15 µl of this DNA solution was hybridized with
PNA Beacon 13mer 100nM with a final volume of 30µl. Before the hybridization with the
beacon, the sample was warmed up to 90°C for 3 minutes and cooled to 50°C for 3 minutes.
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