KR103-Quantscript RT Kit

Quantscript RT Kit
——For first-strand cDNA synthesis and two step RT-PCR

Quantscript RT Kit
Cat. No. 4992783/4992784

Storage
Store at -30~-15°Cfor up to one year.


Introduction
Quantscript RT Kit is designed for the first strand cDNA synthesis from tiny
amount of total RNA or poly(A)+RNA for high sensitive two-step RT-PCR.
Quant Reverse Transcriptase is a new, unique enzyme, different from the
reverse transcriptases of Moloney murine leukemia virus (MMLV) or avian
myeloblastosis virus (AMV). Quant Reverse Transcriptase is a recombinant
enzyme expressed in E. coli. Quant Reverse Transcriptase has a high affinity
for RNA, which enables efficient and sensitive reverse transcription of any
template, leading to high yields of cDNA.

Product Features
This kit includes all relative contents of cDNA first strand synthesis. Quant
Reverse Transcriptase in this kit contains high reverse transcriptase activity.
This product have good performance with RNA templates with high GC
content and complicated secondary structure, have high compatibility
toward downstream PCR or real time PCR and could be combined with any
thermostable polymerase.

Important Notes Before Starting

  1. Solutions used for synthetic reaction of cDNA should be treated with
    0.1% DEPC as far as possible and used after autoclaving. For some
    reagents not suitable for autoclaving, prepare the reagents with
    the sterilized container and water, and then filter to obtain the final
    solutions.
  2. Avoid genomic DNA contaminations for RNA sample.
  3. Repeated freezing and thawing of RNA should be avoided, since this
    leads to reduced RNA quantity.
  4. All the components in the kit should be stored at -30~-15°C.
  5. cDNA synthesized by this kit should be stored at -30~-15°C.

Protocol
The protocol is optimized for use with 50 ng to 2 μg of total RNA. With >2 μg
RNA, scale up the reaction proportionally to the appropriate volume.

  1. Thaw template RNA on ice. Thaw the primer solutions (not supplied),
    10×RT Mix (including RNasin and DTT), Super pure dNTPs (2.5 mM
    each), and RNase-Free ddH2
    O 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 inside walls of the
    tubes.
  2. Prepare a fresh master mix on ice according to Table 1. Mix thoroughly
    and carefully by vortex. Centrifuge briefly to collect residual liquid from
    the inside walls of the tube, and store on ice. Add the RNA template in
    the step 4.
    Note: Set up 10 μl reverse-transcription reaction for downstream Real
    Time qPCR. All the components can be reduced to 1/2 to adapt for
    RealUniversal Color PreMix (4992929/4992881/4992904), SuperReal
    PreMix Plus (4992214/4992215/4992248&4992290/4992291/4992305
    ) and FastFire qPCR PreMix (4992217/4992218/4992249&4992220/49
    92221).
  1. If setting up more than one reverse-transcription reactions, aliquot the
    appropriate volume of master mix into individual reaction tubes. Keep
    tubes on ice.
    Note: A volume of master mix 10% greater than required for the total
    volume of reverse transcription reactions should be prepared.
  2. Add the template RNA (50 ng-2 μg) to the individual tubes containing
    the master mix. Mix thoroughly and carefully by vortex for no more than
    5 sec. Centrifuge briefly to collect residual liquid from the walls of the
    tubes.
  3. Incubate for 60 min at 37°C.
  4. Add an aliquot of the finished RT products to the PCR mix.

KR103-Quantscript RT Kit

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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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