KR118-FastKing gDNA Dispelling RT SuperMix-170801

FastKing gDNA Dispelling RT SuperMix——18 min high-efficient reverse
transcription and gDNA cleaning up in one step

FastKing gDNA Dispelling RT SuperMix
Cat. no. 4992226/4992227/4992251

Storage
Store at -30~-15°C for up to one year

Introduction
FastKing gDNA Dispelling RT SuperMix provides a rapid, stable and efficient
method of cDNA synthesis which is perfect for downstream two-step Real
Time PCR. 5 × FastKing-RT SuperMix contains all the required reagents of
RT-PCR (include FastKing RT Enzyme, RNase Inhibitor, Random primers,
Oligo dT Primer, dNTP Mixture and Reaction Buffer), and a special heatsensitive DNase to efficiently remove genomic DNA without interfering with cDNA. Reaction could be started immediately right after the addition of template RNA and RNase-Free ddH2O.
FastKing RT Enzyme in the SuperMix provides a high-efficient reverse transcription with 42°C, 15 min. With a special modified hydrophobic motif, FastKing Rtase gets a significant affinity for RNA and facilitates the efficiency and speed of the reaction, and enables read-through of templates with high GC content or complex secondary structures.

Product Features
Simple reaction setup: This product is premix-format, the reaction could
be started right after the addition of RNA template and ddH2O.
High performance reverse transcription: 95% of RNA template could be reverse transcribed to cDNA.
Short reaction time:gDNA can be removed and cDNA be synthesized together in one step within 15 min at 42°C.
Good for complex template: This product can be used for the reverse
transcription of RNA template which has complex secondary structure and high GC content.
High compatibility for downstream analysis: This product could be coused with many type of qPCR product for analysis with high sensitivity and stability.

Application Range
RT-PCR; RT-qPCR; cDNA library construction; SAGE

Important Notes

1. This protocol is optimized for use with 50 ng to 2 µg of RNA. With >2 µg
   RNA, scale up the reaction linearly to the appropriate volume.
2. Operate all the experimental process on ice to minimize the risk of RNA
   degradation.
3. Separate denaturation and annealing steps are generally not necessary.
   However, for some RNA templates with complex secondary structure,
   a denaturation step is recommended. If so, denature the RNA before
   reaction setup: incubate the RNA for 5 min at 65°C, then place
   immediately on ice.
4. Reverse transcription system could be scaled up when necessary.

Protocol
Synthesize first-strand cDNA with FastKing gDNA Dispelling RT SuperMix,
this protocol is optimized for the setup of a 20 µl reaction with the RNA
template amount range from 50 ng to 2 μg.

1. Thaw RNA template on ice. Thaw 5 × FastKing-RT SuperMix and RNaseFree ddH2
   O at room temperature (15-30°C), and then place them on ice
   immediately after thawing. Mix each solution by vortex, and centrifuge
   briefly to collect residual liquid from the sides of the tubes.
   Please operate the following steps on ice to guarantee the accuracy
   of reaction setup. Please setup the reaction to mix and then make
   aliquots.
2. Setup the reaction according to the following table.

3. Start the reaction according to the following table.

Note:

1. For downstream qPCR, the volume of reverse transcription product
   added should not exceed 1/10 of the PCR reaction volume. For
   example, add no more than 5 μl of reverse transcription product to a
   50 μl qPCR reaction.
2. Before the downstream qPCR, place the reverse transcription product
   on ice. For longer storage, store the reverse transcription product at
   -30~-15°C.

RNA template quality control
Reverse transcriptase takes RNA as template to synthesize the first strand
cDNA, so the quality of template RNA directly affects the result of reverse
transcription.

1. template integrity: the integrity of template RNA is very important for
   reverse transcription. If RNA template contains RNase, the template
   RNA will be degraded and the amount of cDNA product will be
   decreased or even no cDNA product.
2. template purity: if RNA template contains protein, ions, EDTA, ethanol,
   phenol and other impurities, the activity of the enzyme will be inhibited
   or changed and eventually affects the reverse transcriptional results. If
   genomic DNA is contained, the accuracy of subsequent experiments will
   be affected.
3. template addition: this protocol is optimized for use with 50 ng to
   2 µg of RNA. With >2 µg RNA, scale up the reaction linearly to the
   appropriate volume.

KR118-FastKing gDNA Dispelling RT SuperMix-170801

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This product is for scientific research use only. Do not use in medicine, clinical treatment, food or cosmetics.

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