Direct qPCR ProbesMaster highROX

For research use only.
Please centrifuge briefly before opening (volume ≤2 ml).

Description:
Direct qPCR ProbesMaster highROX is designed for quantitative real-time analysis of target DNA directly from 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 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.

Interference of remaining components from sample matrix
Due to the fast and easy but relatively rough sample preparation remaining components from the sample matrix may be co-transferred into the PCR assay. These remains are mostly blocked by a combination of specially optimized additives. If inhibition of the PCR reaction occurs with higher volumes of transferred sample volume, please reduce the sample volumes or use a dilution of the sample in 1 x Direct Extraction Buffer or water.
Remaining components of the sample matrix may also show fluorescence signals specially in the yellow and red spectral range. If using this fluorescence range for multiplex PCR assays or if using ROX, please take special attention that there is no interference between fluorescence from remaining matrix material and the used fluorescence channels for amplicon detection.

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.

Shipping: shipped on gel packs

Storage Conditions: store dark at -20 °C
avoid freeze/thaw cycles
stable at 4 °C for up to 4 weeks

Shelf Life: 12 months

Form: liquid

Concentration: 2x

Content:
Direct qPCR ProbesMaster highROX
2x conc. mix of antibody-blocked Hot Start polymerase, dNTPs, reaction buffer, 1 μM ROX, additives and stabilizers

Direct Extraction Buffer
5x conc.
Please handle with care and wear personal protective equipment!

PCR-grade Water

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 Blood Samples / Liquid Samples

• Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
• Transfer 2 μl of the Blood/Liquid Sample into a tube containing 100 μl to 200 μl of 1x concentrated Direct Extraction Buffer (a dilution of Blood 1:50 to 1:100 in 1x Direct Extraction Buffer is recommended).
• Close the tube and vortex for 15 sec
• Incubate the tube at room temperature (20-25 °C) for 2-3 min.
• Transfer 1-2 μl of the supernatant into a 20 μl qPCR assay or 2-5 μl into a 50 μl qPCR assay (see table for Preparation of the PCR Assay below).

1.b Samples from nasal or throat swabs

• Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
• Transfer 200 μl 1x Direct 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 1-2 μl of the supernatant into a 20 μl qPCR assay or 2-5 μl into a 50 μl qPCR assay (see table for Preparation of the PCR Assay below).

1.c Samples from Animal or Plant Tissue

• Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
• 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 1x concentrated Direct Extraction Buffer to the tissue sample as following:

• Mix briefly by tapping or vortexing and make sure that the sample is soaked with Direct Extraction Buffer
• Incubate at room temperature (20-25 °C) for 3 min
• Centrifuge briefly and transfer 1-2 μl of the supernatant into a 20 μl qPCR assay or 2-5 μl into a 50 μl qPCR assay (see table for Preparation of the PCR Assay below).

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.

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.

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.

5x Extraction Buffer

Hazard pictograms:

Exclamation mark

Signal word: Warning

Hazard statements:
H315 Causes skin irritation.
H319 Causes serious eye irritation.

Precautionary statements:
P280 Wear protective gloves/protective clothing/eye protection/face protection/hearing protection/....
P302 + P352 IF ON SKIN: Wash with plenty of water/…
P305 + P351 + P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
P321 Specific treatment (see … on this label).
P332 + P313 If skin irritation occurs: Get medical advice/attention.
P362 + P364 Take off contaminated clothing and wash it before reuse.