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Biotechrabbit

[Biotechrabbit] 14X LYO-ready CAPITAL™ qPCR Probe Mix

Cat-No. BC0504305

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14X LYO-ready CAPITAL™ qPCR Probe Mix

 


제품 번호


BC0504305


제품 특징


Features

  • Freeze-dryable formulation including excipients and stabilizers
  • Best-in-class performance in both single and multiplex quantitative amplification
  • Convenient master mix for low-copy number detection
  • High specificity and sensitivity

Applications

  • For use with standard and fast qPCR platforms
  • Single and multiplex qRT-PCR amplification
  • Compatible with a wide range of probe technologies including
  • Taqman®, Molecular Beacons® and Scorpion® probes



biotechrabbit™ 14X LYO-ready CAPITAL™ qPCR Probe Master Mix is a ready-to-use and concentrated formulation of all enzymes, dNTPs, excipients, and stabilizers for producing home-made lyophilizates with the option to include primers and probes. The time-effective lyophilization is possible for high and low volumes of the master mix without the collapse of the lyophilizate. The resulting lyophilizate is highly resistant to humidity and is swiftly dissolved in the provided reconstitution buffer.

The master mix is optimized for real-time PCR quantification of viral DNA, cDNA and genomic DNA from a wide range of targets. The mix ensures high specificity and sensitivity in single and multiplex amplification, making it the best choice for extremely low-copy-number targets in pathogen detection.

CAPITAL qPCR Probe Master Mix uses a proprietary enzyme and buffer formulation suitable for fast extension and multiplexing in challenging PCR reactions.


Component

Composition

14X LYO-ready CAPITAL™ qPCR Probe Master Mix

14X concentrated formulation of a qPCR Probe Master Mix including excipients, ready for lyophilization

4X qPCR Probe Reconstitution Buffer

Optimized PCR reconstitution buffer in 4X concentration

 

Storage

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


Handling
Prevention of contamination

Contamination with undesired DNA is a concern when assembling the amplification reactions. To eliminate the possibility of contamination with undesired DNA, follow the guidelines below:

  • Wear disposable gloves when handling the solutions.
  • Dedicate separate sterile areas for the preparation of samples and reaction mixtures.
  • Use molecular-grade nuclease-free water and reagents.
  • Include a non-template control reaction in every PCR assay.
  • Avoid carryover contamination.
Recommendations before starting a lyophilization project
  • The formulation is optimized for freeze-drying a minimum of 5 reactions of 20 µl.
  • Optional: Prepare concentrated primers and probes in molecular biology grade water and restrict the volume to 20 % of the master mix.
Calculation examples

Three calculation examples for 20 µl reaction size

Parameter

Example A

Example B

Example C

qPCR volume

20 µl

20 µl

20 µl

Reactions per bead/cake

5

50

100

Number of beads/cakes

1

10

50

Total number of reactions

5

500

5000

Total reaction volume

100 µl

10 ml

100 ml

Pipetting scheme

Divide the total reaction volume above by a factor of 14. Minimize the volume of primers and probes.

Component

Example A

Example B

Example C

14X LYO-ready CAPITAL™ qPCR Probe Master Mix

7.1 µl

71 µl

7.1 ml

Optional: Primer & Probes (max. 20 % v/v)

1.4 µl

14 µl

1.4 ml

Volume to freeze-dry per bead/cake

8.5 µl

85 µl

170 µl

Suggested lyophilization protocol

Step

Time*

Temperature

Pressure

Freezing

120 min

−40 °C

—

Primary drying

600 min

−40 °C

0.12 mbar / 12 Pa

Secondary drying

300 min

25 °C

0.01 mbar / 1 Pa

*Duration of freeze-drying cycles depends on the lyophilizate volume, the equipment, and the type and dimensions of the vials.

Reconstitution of lyophilized master mix
  • Add the required volume of the reconstitution buffer to the lyophilized CAPITAL™ qPCR Probe Master Mix.
  • Mix well. The lyophilizate should dissolve swiftly.
  • Store the reconstituted CAPITAL™ qPCR Probe Master Mix at −20 °C.

Parameter

Example A

Example B

Example C

4X Reconstitution Buffer per bead/cake

25 µl

250 µl

500 µl

Total volume of 4X Reconstitution Buffer

25 µl

2.5 ml

25 ml

Cycling program

Step

Temperature

Time

Cycles

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.



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