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

the tubes in which the poly-A-RNA had been fixed. The RNA template is therefore missing in the protocols.

Three different primer pairs were used:

Serotype G: Product app. 800 bp
Serotype D: Product app. 500 bp
Control primer of non-transcribed genomic region: Product app. 400 bp
Amplification reactions were run as duplicates.

One-step RT-PCR

Preparation

Components Volume Final concentration in the reaction dNTP mix (each 10 mM) 1.0 l 200 M Forward primer G (100 M) 1.0 l 2 M Reverse primer D (100 M) 1.0 l 2 M RTplusPCR buffer with 25 mM Mg++ 5.0 l 1 x RNase inhibitor (0.5 g/l) 0.5 l 5 ng/l cMaster RT enzyme (15 U/l) 0.5 l 0.15 U/l cMaster PCR enzyme mix (5 U/l) 0.5 l 0.05 U/l RNase-free water 40.5 l Total volume 50.0 l


Program parameters of the one-step RT-PCR

Step Temp. Duration cDNA synthesis 50C 50 min Initial denaturation 94C 2 min Denaturation 94C 20 s
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IC50: 190 pg/ml · Limit of detection: 60 pg/ml...
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