PCR reactions were performed in a total volume of 25 μL containing 50 ng of DNA, 1 μmol/L of each primer (forward: 5′-ATCTATTGCTGGCACCATCT-3′ and reverse: 5′-CCAATCAAACCCACAGACTTAC-3′), 200 μmol/L dNTPs (Applied Biosystems, Foster City, CA), 2mM MgCl2, 0.25U TaqPol (Applied Biosystems, Foster City, CA, USA) and buffer 1X provided by the manufacturer. PCRs were performed in a 2700 Thermal cycler (Applied Biosystems). The amplifications were done as follows: initial denaturation at 95 °C for 5 min and a final extension at 72 °C for 5 min; denaturation at 95 °C for 30 s, annealing for 30 s at 58 °C, and extension was done at 72 °C for 30 s for 40 cycles. Amplification was verified by gel electrophoresis.
2700 thermal cycler
Manufactured by Thermo Fisher Scientific
The 2700 Thermal Cycler is a laboratory instrument used for the amplification of DNA samples through the process of polymerase chain reaction (PCR). It provides precise temperature control and cycling for the thermal steps required in the PCR procedure.
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2 protocols using 2700 thermal cycler
1
PCR Amplification of DNA Fragments
2
DNA Extraction and PCR Amplification from FFPE Samples
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DNA was extracted from paraffin-embedded tissue samples using the All Prep® DNA/RNA FFPE Kit (50) (Qiagen Cat. No. 80234). PCR experiments were performed in a total volume of 25 µL that contained 50 ng of template DNA, 10 mmol/L Tris-HCl (pH 8.3), 40 mmol/L KCl, 2 mmol/L MgCl2, 200 mmol/L each dNTP, 1 U Platinum Taq DNA Polymerase (Applied Biosystems), and 1 mmol/L each specific primer (forward: 5'-ATCTATTGCTGGCACCATCT-3' and reverse: 5'-CCAATCAAACCCACAGACTTAC-3'). An initial denaturation at 94°C for 5 min was followed by 45 cycles of amplification and a final extension step for 7 min at 72°C. The amplification cycles included denaturation at 94°C for 30 s, 30 s of annealing at 58°C, and 30 s of amplification at 72°C. PCR experiments were carried out in a 2700 Thermal Cycler (Applied Biosystems). The amplification products were verified by agarose gel electrophoresis.
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