Direct qPCR ProbesMaster

Robust real-time PCR master mix for amplification directly from blood, swabs or tissue
2 x conc. master mix.

For in vitro use only!

Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles,
stable at 4 °C for up to 4 weeks
Shelf Life: 12 months
Form: liquid
Concentration: 2 x

Amount

Clear

Price range: $265.00 through $969.00

SKU: N/A Category: Direct qPCR Robust Amplification

Product Information

Direct qPCR ProbesMaster 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

The mix can also be used in combination with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.

Contents:
Direct qPCR ProbesMaster (red cap)
2x conc. mix of antibody-blocked Hot Start polymerase, dNTPs, reaction buffer, 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	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
ROX Reference Dye #PCR-351 ³⁾	25 μM	500 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
³⁾ The mix can be used in combination with ROX reference dye

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 ⁴⁾	15 sec 30-60 sec⁵⁾	35-45x

⁴⁾ The annealing temperature depends on the melting temperature of the primers
⁵⁾ 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.

Use the form below to ask a question or request a quote

Question or Comment

Check the box below if you also need a quote

Need a quote

Additional fields will appear below. We need your address to provide an estimate on shipping costs.

Name

First Last

Email Address(Required)

Address

Street Address Address Line 2 City State / Province / Region ZIP / Postal Code Country

Services

Learn

News

Direct qPCR ProbesMaster

Product Information