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Biotechrabbit

[Biotechrabbit] 4X CAPITAL™ qPCR Probe Master Mix, lyophilized

Cat-No. BR0502601


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4X CAPITAL™ qPCR Probe Master Mix, lyophilized



제품 번호


BR0502601


제품 특징


Features

  • Stable enzyme and mix for ambient shipment and room-temperature storage
  • Best in-class performance for both single and multiplex detection
  • Convenient master mix with high specificity in pathogen detection
  • Highly sensitive for low-abundance DNA targets

Applications

  • Standard and fast cycling qPCR with rapid extension rate for early Ct values
  • For use on a wide range of probe technologies including Taqman®, Molecular Beacons® and Scorpion® probes



Description


biotechrabbit™ lyophilized CAPITAL qPCR Probe Mix is a freeze-dried version of the well-established liquid equivalent. The stabilized format allows shipment and storage without cooling. The master mix is optimized for quantifying genomic, cDNA and viral sequences provides outstanding performance in single and multiplex qPCR. The high sensitivity provided by the mix is ideal for detection of low-abundance DNA targets in various applications, such as pathogen detection. CAPITAL qPCR Probe Mix uses proprietary combination of enzyme and buffer chemistry for efficient extension and early Ct in single and multiplex qPCR.

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



Other Information


Component

Composition

Lyo CAPITAL qPCR Probe Mix

Cake of lyophilized 4× qPCR Probe Master Mix

qPCR Probe Reconstitution Buffer

Optimized PCR buffer for reconstituting lyophilized CAPITAL qPCR Probe Mix

 

LYO Master Mix RECONSTITUTION

1) Transfer the whole content of one vial qPCR Mix Reconstitution Buffer to one vial Lyo CAPITAL qPCR Probe Mix
2) Mix well – the lyophilisate will dissolve within seconds
3) Store the reconstituted CAPITAL qPCR Probe Mix at -20°C 

STORAGE

Store at room temperature or below (until expiry date – see product label)
Reconstituted lyophilisate: store at −20°C for up to 12 months

 

 

Quality Control
Functional assay

Mix tested functionally in qPCR.



Protocols

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.
  • Amplicon lengths should not exceed 400 bp.
  • 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 PCR contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

  • Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
  • Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
  • Use only water and reagents that are free of DNA and nucleases.
  • With every PCR setup, perform a contamination control reaction that does not include 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 DNA

Variable

10 pg – 100 ng

Use diluted or undiluted cDNA from less than 1 µg RNA

CAPITAL qPCR Probe Mix, 4×
(reconstituted lyophilisate)

5 µl

1×

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

Initial activation

95°C

2-3 min

1

Denaturation

95°C

10–15 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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