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CAPITAL™ qRT-PCR Probe Mix, 4×

Features

  • Best in-class performance for both single and multiplex detection
  • Convenient master mix for detection of low-copy pathogen targets
  • High specificity and sensitivity across a wide range of sample sources

Applications

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

Purchase CAPITAL™ qRT-PCR Probe Mix, 4×

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

Catalog # Size Package Content Price* Qty
BR0502001 200 rxn of 20 µl 1 ml CAPITAL qPCR Probe Mix (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502002 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Probe Mix (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502003 4000 rxn of 20 µl 4 kits BR0502002 CAPITAL qRT-PCR Probe Mix (1000 rxn each)
€1,966.00
BR0502101 200 rxn of 20 µl 1 ml CAPITAL qPCR Probe Mix LRox (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502102 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Probe Mix LRox (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502103 4000 rxn of 20 µl 4 kits BR0502002 CAPITAL qRT-PCR Probe Mix LRox (1000 rxn each)
€1,966.00
BR0502201 200 rxn of 20 µl 1 ml CAPITAL qPCR Probe Mix HRox (1step)
200 µl RTase with RNase Inhibitor
€138.00
BR0502202 1000 rxn of 20 µl 5 × 1 ml CAPITAL qPCR Probe Mix HRox (1step)
5 × 200 µl RTase with RNase Inhibitor
€556.00
BR0502203 4000 rxn of 20 µl 4 kits BR0502202 CAPITAL qRT-PCR Probe Mix HRox (1000 rxn each)
€1,966.00

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

OR

biotechrabbit CAPITAL qRT-PCR Probe Mix provides outstanding performance for real-time PCR quantification of RNA templates, including mRNA, total RNA and viral RNA from a wide range of targets. The master mix ensures high specificity and sensitivity in single and multiplex detection, making it the choice for extremely low-copy-number targets in pathogen detection.

CAPITAL qRT-PCR Probe 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 concentrations.

 

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

 

Component

Composition

CAPITAL qPCR Probe Mix (1step)

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

 LRox / HRox mixes

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/).
  • For TaqMan® probes choose probe close to 5’ primer, avoid terminal guanosine residues.
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.

Specific Probe

Variable

200 nM

Template RNA

Variable

0.01 pg to 1 µg

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

CAPITAL qPCR Probe 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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