Manual

Product Number: PC58             for qPCR First Strand cDNA Synthesis Kit

Storage

Store at -20℃, valid for 12 months.

Components

Component	PC5801 50rxns	PC5802 100rxns
5×RT PCR MasterMix	200 μl	400 μl
RNase free H2O	1 ml	2×1 ml

Description

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.

Application

First Strand cDNA Synthesis.

Detecting high copy and low copy genes.

Features

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

Protocol

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.

Components	Volume
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.

RT-qPCR

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.

Note

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.