Description:
Direct qPCR ProbesMaster highROX is designed for quantitative real-time analysis of target DNA directly from whole blood, swabs and animal- or plant tissue. The mix allows robust amplification avoiding the requirement of any prior DNA purification procedures.
The mix is recommended for use with dual-labeled fluorescent probes, e.g. TaqMan®, Molecular Beacon or Scorpion probes. It provides a powerful tool for multiplex-quantification of sample DNA in a broad dynamic range with exceptional sensitivity and precision.
The mix contains all reagents required for qPCR (except template, primer and labeled fluorescent probe) in a premixed 2 x concentrated ready-to-use solution. High robustness, reliability and sensitivity of the mix are based on a an antibody-blocked hot start polymerase in combination with an optimized and well-balanced buffer system.
The mix ensures fast and easy preparation with a minimum of pipetting steps and is specially recommended for:
- Direct detection of viral or bacterial DNA in nasal or throat swabs
- Direct PCR from whole blood samples
- Direct amplification of target DNA from various tissue samples
- Point-of-Care diagnostics.
Multiplexing Capability
Real-time PCR technology based on dual-labeled DNA probes provides a high sensitive and specific PCR system with multiplexing capability. The simultaneous detection of multiple targets in a single tube requires a primer/probe set for each target amplification. Sequences and concentrations of primers and probes should be optimized to avoid mutual influence, secondary structures and primer-dimer formations. Amplification of each target is detected in a separate fluorescence channel.
ROX reference dye
Direct qPCR ProbesMaster highROX contains 500 nM ROX passive reference dye in the final assay. The dye does not take part in the PCR reaction but allows to normalize for non-PCR related signal variation and provides a baseline in multiplex reactions.
The reaction chemistry of the kit is optimized for instruments that are compatible with the evaluation of a high ROX reference signal.
Contents:
Direct qPCR ProbesMaster highROX (red cap)
2x conc. mix of antibody-blocked Hot Start polymerase, dNTPs, reaction buffer, 1 μM ROX, additives and stabilizers
Extraction Buffer (yellow cap)
10x conc.
Please handle with care and wear personal protective equipment!
PCR-grade Water (white cap)
Procedure
Before starting, take reagents out from fridge and allow to thaw completely. Vortex all reagents briefly and spin down the liquids.
1. Sample preparation
1.a Whole Blood (not recommended for heparin-, EDTA- or citrate-treated whole blood)
- Add whole blood (1-2 μl for 20 μl or 2-5 μl for 50 μl total assay volume) without any pre-treatment directly to the qPCR assay.
1.b Samples from nasal or throat swabs
- Dilute 10x Extraction buffer to 1x with PCR-grade water
- Transfer 200 μl 1x Extraction Buffer into a 1.5 ml microtube
- Cut off the cotton tip with the collected nasal or throat swab and place it in the micro tube
- Close the tube and vortex for 15 sec
- Incubate at room temperature (20-25 °C) for 2-3 min
- Remove the cotton tip and squeeze it out at the rim of the tube
- Centrifuge briefly and transfer 1-5 μl of the supernatant (1-2 μl for 20 μl or 2-5 μl for 50 μl total assay volume) to the qPCR assay.
1.c Samples from Animal or Plant Tissue
- Prepare a small piece from animal or plant tissue not exceeding 8 mm in diameter
- Crack plant seeds to less than 1 mm in diameter using a BeadBeater, Tissue Lyser or small hammer
- Place the sample in a 1.5 ml microtube
- Add Extraction Buffer to the tissue sample as following:
| Sample size (diameter) |
1-2 mm |
3-4 mm |
5-8 mm |
| PCR-grade water |
45 μl |
90 μl |
180 μl |
| Extraction Buffer |
5 μl |
10 μl |
20 μl |
- Mix briefly by tapping or vortexing and make sure that the sample is soaked with Extraction Buffer
- Incubate at room temperature (20-25 °C) for 3 min
- Centrifuge briefly and transfer 1-5 μl of the supernatant (1-2 μl for 20 μl or 2-5 μl for 50 μl total assay volume) to the RT-qPCR assay
- If sample is liquid: Dilute 10x Extraction buffer to 2x with PCR-grade water. Add 2x Extraction buffer to your sample in a ratio of 1:1.
2. Preparation of the PCR Assay
Preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified below. A reaction volume of 20-50 μl is recommended for most real-time instruments. Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
| component |
stock conc. |
final conc. |
20 μl
assay |
50 μl
assay |
| Direct qPCR ProbesMaster highROX |
2x |
1x |
10 μl |
25 μl |
| Extracted Sample or whole blood |
- |
- |
1-2 μl |
2-5 μl |
| Forward Primer 11) |
10 μM |
300 nM |
0.6 μl |
1.5 μl |
| Reverse Primer 11) |
10 μM |
300 nM |
0.6 μl |
1.5 μl |
| TaqMan® / Dual Labeled Probe 11) |
10 μM |
200 nM |
0.4 μl |
1 μl |
| Forward Primer 22) |
10 μM |
300 nM |
0.6 μl |
1.5 μl |
| Reverse Primer 22) |
10 μM |
300 nM |
0.6 μl |
1.5 μl |
| TaqMan® / Dual Labeled Probe 22) |
10 μM |
200 nM |
0.4 μl |
1 μl |
| PCR-grade water |
- |
- |
fill up to
20 μl |
fill up to
50 μl |
1) The optimal concentration for primers and probe may vary from 100 to 500 nM and should be optimized for each new assay set-up
2) Required only for multiplex PCR applications
Mix the tubes briefly and spin down to remove bubbles.
3. PCR Cycling
Switch on the real-time PCR cycler and set all cycling parameters as recommended in the table below. Place the vials into the instrument and start the program.
Initial
denaturation |
95 °C |
2 min |
1x |
Denaturation
Annealing and Elongation |
95 °C
60-65 °C 3) |
15 sec
30-60 sec4) |
35-45x |
3) The annealing temperature depends on the melting temperature of the primers
4) The elongation time depends on the length of the amplicon. A time of 30 sec is sufficient for fragments < 500 bp
To obtain optimal specificity and amplification results an individual optimization of the recommended parameters is recommended for each particular sample/primer pair.
4. Data Analysis
- Calculate ct-values and evaluate the data according to the instruction of the cycler and requirements of the experiment/application.
