Product Number: PC58 for qPCR First Strand cDNA Synthesis Kit
Store at -20℃, valid for 12 months.
|Size||20 μl × 50 rxns||20 μl × 100 rxns|
|5×RT PCR MasterMix||200 μl||400 μl|
|RNase free H2O||1 ml||2×1 ml|
The 5×RT PCR MasterMix offer a whole component for cDNA synthesis. Which is a complete system for the efficient synthesis of first strand cDNA from RNA templates. The Mix utilizes the recombinant thermostable M-MuLV Reverse Transcriptase which exhibits lower RNase H activity than AMV reverse transcriptase.
This MasterMix already include all the reaction component. the thermostable M-MuLV Reverse Transcriptase, RNase Inhibitor, Random primers, Oligo dT Primer, dNTP Mixture, Buffer. When set up the reaction only need add water and RNA template.
First Strand cDNA Synthesis.
Detecting high copy and low copy genes.
1. Themo stable MMLV, high efficiency and stable.
2. Whole component Mix, simple for reaction setup, fast reaction.
3. High concentration, much more volume for RNA template, suitable for low copy RNA template amplification.
4. The premix is not frozen under-20℃, simplified the Thaw and mixing progress.
5. Specially optimize for qPCR
Reaction set up
First Strand cDNA Synthesis (20μl reaction as a sample, 10μl also available)
1. Template RNA thawed on ice. RNase free H2O thawed under RT and put on ice immediately.
2. Add the flowing component in a RNase free PCR tube.
|Total RNA/mRNA||≤ 16 μl *|
|5×RT PCR MasterMix||4 μl** (See Note 3)|
|RNase free H2O||to 20 μl|
* Total RNA no more than 2 μg, mRNA no more than 200 ng (20μl reaction)
3. Gently mix the solution (total 20 μl)
If mRNA template source from eucaryotic cell (e.g., human mice cell) which has Poly(A) Tail, 42℃ Incubation 15 min.
If mRNA Template source from bacterial or virus prokaryotic cell, do not have Poly(A) tail, 25℃ Incubation 10 min, 42℃ Incubation 15 min.
Mark: If template has Complex secondary structure or GC rich can set up reaction temperature at 50℃.
4. Inactivation Reverse Transcriptase under 85℃ for 5 sec.
5. The obtained cDNA products can be immediately used for qPCR reaction, or stored at -20 ℃ and used within six months; Long term storage is recommended to be stored at -70 ℃ after packaging. CDNA should avoid repeated freeze-thaw.
Take a suitable amount of cDNA products (generally not more than 1/10 of the volume of qPCR reaction) as the template of qPCR. If the expressed genes are abundant, they can be diluted according to actual conditions.
1. Avoid of RNase pollution
2. To make sure reaction successful, the high-quality RNA template is suggested.
3. ** 5×RT PCR MasterMix is Very viscous, and easily adsorbed on the tube and pipe tips causing loss. Pleas centrifuge shortly before use. The enzyme in the 5×RT PCR MasterMix are all excessive 3.6 μl-3.8 μl per prep will not affect the reaction.