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CAPITAL™ qRT-PCR Green Mix, 4×

Features

  • Convenient mix for quantification of RNA templates
  • Sensitive and specific amplification with rapid extension rate for early Ct values
  • Excellent linearity across a wide range of RNA dilution

Applications

  • One step qRT-PCR from mRNA, total RNA and viral RNA targets
  • For use with standard and fast qPCR platforms

Purchase CAPITAL™ qRT-PCR Green Mix, 4×

Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

Catalog # Size Package Content Price* Qty
BR0502301 200 rxn of 20 µl 1 ml CAPITAL qPCR Green Mix (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502302 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Green Mix (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502303 4000 rxn of 20 µl 4 kits BR0502302 CAPITAL qRT-PCR Green Mix (1000 rxn each)
€1,966.00
BR0502401 200 rxn of 20 µl 1 ml CAPITAL qPCR Green Mix LRox (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502402 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Green Mix LRox (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502403 4000 rxn of 20 µl 4 kits BR0502402 CAPITAL qRT-PCR Green Mix LRox (1000 rxn each)
€1,966.00
BR0502501 200 rxn of 20 µl 1 ml CAPITAL qPCR Green Mix HRox (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502502 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Green Mix HRox (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502503 4000 rxn of 20 µl 4 kits BR0502502 CAPITAL qRT-PCR Green Mix HRox (1000 rxn each)
€1,966.00

*Does not include applicable tax, shipping & handling, or other charges.

OR

biotechrabbit CAPITAL™ qRT-PCR Green Mix allows sensitive and specific cDNA synthesis and qPCR in a single tube for quantifying mRNA, total RNA and viral RNA sequences. Extremely low-copy-number targets can be detected with high efficiency over several logs of template concentration.

CAPITAL qRT-PCR Green Mix uses proprietary reverse transcriptase technology and buffer chemistry for efficient cDNA synthesis and QPCR in a single tube. To enable the use of the kit on qPCR platforms with different reference dye concentration requirements, three kit formats are available: a one-step kit containing no ROX, as well as LRox and HRox versions containing ROX in the corresponding concentratios.

 

Info: Recommended annealing temperature is 2°C above primer Tm (use gradient PCR to optimize the annealing temperature).

 

Component

Composition

CAPITAL qPCR Green Mix (1step)

Optimized 4× qPCR Green Master Mix for One Step qRT-PCR

LRox / HRox mix

Rox incorporated in the mix in low / high concentration 

RTase with RNase Inhibitor

Proprietary 20× Reverse transcriptase in a mix with efficient Ribonuclease Inhibitor

 

STORAGE

−20°C (until expiry date – see product label).

 

Quality Control
Functional assay

Mix tested functionally in QRT-PCR.

 
ROX reference dye
  • See PCR cycler instruction for recommended concentration of ROX passive reference dye.
Notes
  • For efficient amplification under fast cycling conditions use amplicon lengths between 80 bp and 200 bp.
  • The shorter the amplicon length the faster the reaction can be cycled. Use maximum 400 bp amplicons.
  • Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings (http://frodo.wi.mit.edu/primer3/).
Prevention of reaction contamination

RNase contamination is an exceptional concern when working with RNA. RNase A, providing most threat to RNA integrity, is a highly stable contaminant of any laboratory. To prevent RNA from degradation and to minimize possibility of contamination One Step RT-PCR; follow the guidelines below:

  • Use separate clean areas for preparation of the samples and the reaction mixture.
  • DEPC-treat all tubes and pipette tips or use certified nuclease-free labware with aerosol filters.
  • Wear fresh gloves when handling RNA and all reagents.
  • Always assess the integrity of RNA prior to RT-PCR in denaturing agarose gel electrophoresis.
  • Use only water and reagents that are free of DNA, DNases and RNases.
  • With every One Step RT-PCR setup, perform a contamination control reaction without template DNA.
Basic Protocol
  • Keep the master mix protected from light until you use it.
  • Aliquot the master mix to minimize freeze-thaw cycles and light exposure.
  • Thaw on ice and mix very well all reagents. Assemble and keep all reactions on ice.
  • Use only high quality optically clear reaction plates and seals designed for fluorescence applications.
  • Do not use corner wells or use a more robust seal.
  • Reserve plate positions for positive (control DNA) and negative (water or buffer) controls.
  • First pipette the primer mixture, then add the template and last the Master Mix.
  • Before preparing mixes, calculate the volume needed according to the reaction number plus one extra.
  • To have a better correlation, run the reactions in triplets.

Component

Volume

Final concentration

Primer Mix (Reverse and Forward)

Variable

100–400 nM

Too high primer concentrations result in unspecific amplification and should be avoided.

Template RNA

Variable

0.01 pg to 1 µg

Use 1 pg – 1 µg Total RNA, or >0.01 pg mRNA

CAPITAL qPCR Green Mix (1step), 4×

5 µl

RTase with RNase Inhibitor, 20×

1 µl

Nuclease free water

Variable

 

Total volume

20 µl

 

  • Gently mix the reactions without creating bubbles (do not vortex). Bubbles will interfere with fluorescence detection. Place the reaction into the PCR cycler.
Cycling Program

Step

Temperature

Time

Cycles

Reverse Transcription

50°C

10 min

1

Initial activation

95°C

3 min

1

Denaturation

95°C

10 s

40-45

Annealing/Extension*

(60-68°C)

30 s

*Recommended annealing/extension temperature is primer Tm +2°C. Use gradient PCR to optimize the annealing temperature. Do not use temperatures below 60°C. Do not exceed 30 seconds.
For melt analysis refer to instrument instructions. 

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