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Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf ,,, cMaster RTplusPCR System

Annealing 45C 20 s 40x Elongation 68C 30 s Cooling 4C Hold
Two-step RT-PCR

Gene-specific primers were used for the cDNA synthesis.

Reverse transcription preparation

Components
Volume Final concentration in the reaction dNTP mix (each 10 mM) 2.0 l 1 mM Reverse primer G (100 M) 1.0 l 5 M Reverse primer D (100 M) 1.0 l 5 M Left control primer (100 M) 1.0 l 5 M RNase-free water 5.0 l Master mix 1 10.0 l RTplusPCR buffer with 25 mM Mg++ 4.0 l 2 x RNase inhibitor (0.5 g/l) 0.5 l 12.5 ng/l cMaster RT enzyme (15 U/l) 2.0 l 1.5 U/l RNase-free water 3.5 l Master mix 2 10.0 l
Program parameters of the reverse transcription

Step Temp. Duration Template RNA denaturation 65C
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