Fisrt strand cDNA Synthesis KIT（反轉錄合成試劑盒）是專為兩步RT-PCR 第一步實驗配製的，具有高靈敏度的RT-PCR反應系統，可以從極低量的總RNA 或poly(A)+ RNA 合成cDNA。與PCR 反應相結合，可用於檢測稀有基因的表達、從極少量細胞中定量檢測特定mRNA 的表現量、clone特定基因的cDNA 片段等。

First Strand cDNA Synthesis Kit 25 rxns
 
 M-MuLV Reverse Transcriptase (20 U*/µL) 60 µL
 RNase Inhibitor (20 U**/µL) 30 µL
 5X Reaction Buffer  125 µL
 10mM dNTP Mix 65 µL
 Oligo(dT)18 Primer 100 µM, 0.5 µg/µL  30 µL
 Random Hexamer Primer 100 µM, 0.2 µg/µL 30 µL
 Water, nuclease-free 650 µL
 
PROTOCOLS
RT-PCR I. First Strand cDNA Synthesis After thawing, mix and briefly centrifuge the components of the kit. Store on ice.
 
1. Add the following reagents into a sterile, nuclease-free tube on ice in the indicated order: Template RNA total RNA (0.1 - 5 µg) or poly(A) mRNA  (10 ng - 0.5 µg) or specific RNA( 0.01 pg - 0.5 µg ) Primer oligo (dT)18 primer or random hexamer primer or gene-specific primer 1 µL 1 µL 15-20 pmol Water, nuclease-free to 11 µL Total volume 11 µL
2.Optional. If the RNA template is GC-rich or contains secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min. Chill on ice, spin down and place the vial back on ice.
3. Add the following components in the indicated order: 5X Reaction Buffer 4 µL RiboLock RNase Inhibitor (20 U/µL) 1 µL 10 mM dNTP Mix 2 µL M-MuLV Reverse Transcriptase (20 U/µL) 2 µL Total volume 20 µL
4.Mix gently and centrifuge.
5. For oligo(dT)18 or gene-specific primed cDNA synthesis, incubate for 60 min at 37°C. For random hexamer primed synthesis, incubate for 5 min at 25°C followed by 60 min at 37°C. Note. For GC-rich RNA templates the reaction temperature can be increased up to 45°C.
6. Terminate the reaction by heating at 70°C for 5 min. The reverse transcription reaction product can be directly used in PCR applications or stored at -20°C for less than one week. For longer storage, -70°C is recommended.