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First cycle, first strand cDNA synthesis
The Marathon cDNA Amplification Kit method employs a specially-designed adaptor that significantly reduces background and permits both 5'- and 3'-RACE reactions (1, 2) to be performed using the same template. Marathon cDNA amplification can be used to quickly characterize multiple RNAs identified by expressed sequence tags (ESTs), differential display, RNA fingerprinting, or cDNA subtraction. Marathon cDNA synthesis begins with poly A+ RNA and a modified lock-docking oligo(dT) primer that contains two degenerate nucleotides at the 3' end. These nucleotides position the primer at the beginning of the poly A+ tail, eliminating the 3' heterogeneity inherent with conventional oligo(dT) priming. Following cDNA synthesis, blunt ends are created and the Marathon Adaptor is ligated to both ends of the double-stranded cDNA.
The RevertAid First Strand cDNA Synthesis Kit contains RevertAid Reverse Transcriptase, RiboLock RNase Inhibitor, 5X Reaction Buffer, dNTP Mix, Oligo(dT)18 Primer, Random Hexamer Primer, Control GAPDH RNA, 10 μM Forward GAPDH Primer, 10 μM Reverse GAPDH Primer, and nuclease-free water.
Store at -20°C.
Second Cycle, first strand cDNA synthesis
We tested the effect of T4gp32 on the first strand synthesis of A. thaliana mRNA using three alternative protocols. First, we reverse transcribed with Superscript II for 40 min at 45°C, in the absence (RT1) versus the presence (RT2) of T4gp32. Figure 4A shows the unbound and incomplete cDNA (−) fractions, as compared with the bound and full-length cDNA (+) fractions, for each RT condition. These results are visualized in the histogram presented in Figure 4B, which shows the RT efficiency for each RT condition and the percentage of first-strand cDNA that was CAP trapped. The presence of T4gp32 had very little effect on the overall RT efficiency (RT1: 19.6% without T4gp32; RT2: 21.2% with T4gp32), but improved drastically the amount of full-length cDNA that was CAP trapped (from 26.6% without to 44.3% with T4gp32).
QuantiTect Reverse Transcription Kit - QIAGEN
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