KR116-FastKing RT Kit (With gDNase)-180123

FastKing RT Kit (With gDNase)——21 min high-efficient reverse
transcription with gDNA cleaning up

FastKing RT Kit (With gDNase)
Cat. no. 4992223/4992224/4992250

Introduction
FastKing RT kit is an efficient, rapid and genomic DNA cleaning up reverse
transcription system. This product contains gDNase which can remove
genomic DNA by incubation at 42°C, 3 min to protect the total RNA from
genomic DNA interference. King RT Enzyme in RT Enzyme Mix provides
a high-efficient reverse transcription with 42°C, 15 min. With a special
modified hydrophobic motif, King RTase gets a significant affinity for
RNA and facilitates transcription through of RNA templates, and enables
read-through of templates with high GC content or complex secondary
structures.

Product Features
High RT efficiency: RT efficiency higher than 95%
Simple and easy to operate: Simple reaction set up, first strand cDNA can
be synthesis within 21 minutes.
Read complex template: Enables read-through of templates with high GC
content or complex secondary structures.
High sample universality: Cabable for high impurity content RNA
templates and RNA templates with different species.
Excellent capability: Could be co-used with qPCR product with high
sensitivity and stability.

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 on ice to minimize the risk of RNA degradation.
3. Separate denaturation and annealing steps are generally not necessary.
   However, for some RNAs with a high degree of secondary structure, a
   denaturation step may be desired. If so, denature the RNA in RNase-free
   water before reaction setup: incubate the RNA for 5 min at 65°C, then
   place immediately on ice.
4. For some transcripts, oligo-dT primers or Gene Specific Primer is
   possible. The final primer concentrations are as follow: Oligo-dT Primer
   50 pmol/20 μl reaction system; Gene Specific Primer 5 pmol/20 μl
   reaction system.
5. When using Gene Specific Primers, we recommend a reverse
   transcription temperature of 42°C, 15 min. Raise the reaction
   temperature to 50°C will be helpful when non-specific amplification
   appeared.
6. Reverse transcription system could be scale up when necessary.

Protocol
The protocol is optimized for use with 50 ng to 2 μg of RNA.

1. Thaw template RNA on ice. Thaw 5 × gDNA Buffer, FQ-RT Primer Mix,
   10×King RT Buffer, RNase-Free ddH2O at room temperature (15-30°C).
   Place on ice immediately after thawing. Mix each solution by vortex,
   and centrifuge briefly to collect residual liquid from the sides of the tubes.
   The following steps are all requires operate on ice to guarantee the
   precision of reaction set up. Reagent mix should be set up before
   every reaction, then aliquot the mix to each tube.
2. Prepare a fresh master mix to clean up genomic DNA according to Table，Mix thoroughly and carefully for no more than 5 sec. Centrifuge briefly to collect residual liquid from the sides of the tube, incubate the mixture for 3 min at 42°C then store on ice.

3. Prepare a fresh master mix for reverse transcription according to table 2.
   Mix thoroughly and carefully for no more than 5 sec. Centrifuge briefly
   to collect residual liquid from the walls of the tube.

4. Add reverse transcript mixture into the liquid getting from step 2, mix
   thoroughly.
5. Incubate for 15 min at 42°C.
6. Incubate for 3 min at 95°C, then put on ice. The cDNA could be used in
   following experiments or stored in low temperature.

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.

KR116-FastKing RT Kit (With gDNase)-180123

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