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TonkBio™ First Chain cDNA Synthesis Kit 

The TonkBio™ First Chain cDNA Synthesis Kit for RT-PCR is optimized to efficiently synthesize first cDNA from mRNA or total RNA templates.
Cat. No. Size Price Quantity  
TB30001A 50 rxns USD 120
TB30001B 100 rxns USD 190
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

TonkBio™ M-MLV (200 U/ μL)
OligodT primer
Random primer
5 × Reaction buffer
dNTP mix (10 mM each)
RNase Inhibitor
RNase-free H2O

Storage and Stability

Store all system components at -20℃ for one year.

Description

The TonkBio™ First Chain cDNA Synthesis Kit for RT-PCR is optimized to efficiently synthesize first chain cDNA from mRNA or total RNA templates. The kit uses M-MLV reverse transcriptase with lower RNase H activity, which avoids degradation of RNA when RNA-DNA hybrid forms in the progress of cDNA synthesis. It is suitable for synthesis of cDNA up to 9 kb.
The cDNA products could be used directly in a variety of downstream applications, including PCR, qPCR, gene clone.

Caution

  1. For research use only
  2. Use RNase-free reagents during RNA extraction and cDNA synthesis process.
  3. NO repeated freezing and thawing RNA. RNA should be dissolved in ice-water mixture.
  4. The reverse transcription reaction products should be stored at -20℃.
  5. Wear gloves when handling RNA and all reagents as skin is a common source of RNases.

Synthesis

Standard cDNA Synthesis

1. Fisrt-chain cDNA synthesis

Store the components of the kit on ice after thawing, mixing and brief centrifugation.

1) Add the following components into a sterile, nuclease-free tube on ice:

Total RNA / mRNA 0.01 -5 μg/ 10 pg-0.5 μg
OligodT primer / Random primer 1 μL / 20 pmol
RNase free H2O up to 12.5 μL

2) Incubate at 65℃ for 5 minutes. Chill on ice, spin down and place the vial back on ice.

3) Add the following components:

5 × Reaction buffer 4 μL
RNase Inhibitor 0.5 μL
dNTP mix (10 mM each) 2 μL
TonkBio™ M-MLV (200 U/ μL) 1 μL

Mix gently and centrifuge briefly.

4) For a reaction using OligodT or gene specific primers, incubate at 42℃ for 1 hour. For random hexamer primed synthesis incubate at 25℃ for 5 minutes followed by 42℃ for 1 hour.

5) Terminate the reaction by heating at 70℃ for 5 minutes.

*All operations must be done on ice.

Time of reverse transcription can be adjusted at 30-60 minutes. And extending the reverse transcription time is helpful in obtaining longer cDNA.

2. PCR Amplification of First Chain cDNA

The product of the first chain cDNA can be used directly in PCR or qPCR. The volume of the first strand cDNA synthesis reaction mixture should be no more than 1/10 of the total PCR reaction volume. For qPCR amplification, the amount of the total RNA is no more than 1μg.

PCR system recommended:

the first-chain cDNA 1-2 μL
Primer-F (10 μM) 1 μL
Primer-R (10 μM) 1 μL
2 PCR Master Mix 25 μL
ddH2O up to 50 μL

Troubleshooting

Troubleshooting Guide

1. NO or low product of RT PCR

1) Degradation of RNA template. RNA Purity and integrity is important to obtain full length cDNA. Users have to make gel electrophoresis detection to ensure no degradation occurred. And avoid repeated freezing and thawing RNA.

2) Low concentration of RNA template. Trace amount of agents used in RNA purification may remain in solution and inhibit cDNA synthesis. Suggest to wash RNA with 75% ethanol ( prepared by DEPC water) carefully.

3) Incorrect primers. Select random primers or specific primers instead of OligodT primer with bacterial RNA or RNA without a poly A tail.

4) GC rich template. If the RNA template is GC-rich or contains secondary structures, choose random primers and increase the reaction temperature of transcription up to 55℃.

2. RT-PCR product longer than expected

RNA sample is contaminated with DNA. To avoid genomic DNA contamination, User could use DNase I to digest RNA sample before transcription or design primers on exon-intron boundaries.

3. Product of RT-PCR in negative control

RNA template is contaminated with DNA. Perform DNaseI digestion prior reverse transcription.

Datasheet Download

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