sign inorregister |Items in Cart ( )

Products

Home > RT-PCR > TonkBio™ RT Reagent Kit (for qPCR)

TonkBio™ RT Reagent Kit (for qPCR) 

The TonkBio™ RT reagent Kit is designed for the first step of RT-PCR. TonkBio™ M-MLV with high extension ability can complete cDNA synthesis in 15 minutes. This kit is suitable for high throughput analysis because its easy operation and short time consumed.
Cat. No. Size Price Quantity  
TB30003A 100 rxns USD 190
TB30003B 200 rxns USD 320
Tel: +1 (973) 789-8318Email: [email protected]

Description

The TonkBio™ RT reagent Kit is designed for the first step of RT-PCR. TonkBio™ M-MLV with high extension ability can complete cDNA synthesis in 15 minutes. This kit is suitable for high throughput analysis because its easy operation and short time consumed.

The TonkBio™ RT reagent Kit supplies two kinds primers, the OligodT primer and Random primer. Choose correct primers according to specific reaction. The components of this kit have high compatibility with qPCR.

Reagents Supplied

TonkBio™ RT Enzyme Mix 100 μL
OligodT primer (50 μM) 100 μL
Random primer (100 μM) 400 μL
5 × Reaction buffer (for qPCR) 400 μL
RNase-free H2O 1.2 mL

TonkBio™ RT Enzyme Mix contains RNase inhibitor.
5 × Reaction buffer contains dNTP and Mg2+.

Storage and Stability

Store all system components at -20°C for one year.

Caution

  1. Used for research only.
  2. To avoid error and waste, user could prepare the reaction reagent mixture and then divide it into several reaction tubes.
  3. Centrifuge briefly before using TonkBio™ RT Enzyme Mix.

Transcription

Standard of Transcription

After thawing, mix and briefly centrifuge the components of the kit. Store on ice.

Add the following components in RNase-free tube as shown (operate in ice):

RNA template 1 pg- 1 μg *
5 × Reaction buffer 2 μL
TonkBio™ RT Enzyme Mix 0.5 μL
OligodT primer 0.5 μL
Random primer 0.5 μL
RNase free H2O add to 10 μL

Mix gently and centrifuge briefly.

  1. Incubate at 42°C for 15 minutes.
  2. Terminate the reaction by heating at 85°C for 5 seconds.

* User should adjust the amount of RNA according to level of gene expression. Usually, 1 pg-1 μg total RNA is enough for a volume of 10 μL reaction. Because the amount of RNA participated in transcription changes as RNA types and quality, user could change the amount of the RNA. Also, the reaction system can be enlarged if necessary.

Using OligodT primer and Random primer together obtain the full length cDNA efficiently. Also, User can use them separately.

If the specific primer want to be used in transcription, 1 pmol of the specific primer is suggested. And increasing the temperature during the transcription will avoid nonspecific amplification.

The cDNA products can be stored at -20°C or used to PCR amplification directly.

PCR Amplification of First Strand cDNA

The volume of the first strand cDNA synthesis reaction mixture should not more than 1/10 of the total PCR reaction volume. Normally, 1 μL the cDNA synthesis reaction mixture is used as template for subsequent PCR in 25 μL total volume.

PCR system recommended:

the first-strand cDNA 1 μL
Primer-F (10 μM) 1 μL
Primer-R (10 μM) 1 μL
2 × Golden SYBR Green qPCR Mix 12.5 μL
ddH2O up to 25 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 RNA should avoid repeated freezing and thawing.

2) Low concentration of RNA template. Trace the 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°C.

2. Genomic DNA in RNA sample

To avoid genomic DNA contamination, User could use DNase I to digest RNA sample before transcription or design primers.

3. Product of RT-PCR in negative control

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

Datasheet Download

Send us an inquiry

Our teams will reply to your inquiry within 2 hours or you can call us with any requirements and feedback.
Your information is safe with us.

  • * Indicates a Required Field
  • * Name cannot be empty
  • * Provide a valid email address
  • * Comments cannot be empty
  • Submit