lyophilised RT-real-time-PCR Master Mix for dual labeled probes
For general laboratory use.
Shipping: shipped at ambient temperature
Storage Conditions: store at ambient temperature
Store in an aluminium-coated bag or on a dry place.
Lyophilisates may hydrate at humidity levels >70 % when sealing is opened.
Shelf Life: 6 months in sealed package
SCRIPT RT-qPCR ProbesMaster Lyophilisate is designed for performing maximum sensitive and specific RT-qPCRs convenient in single tubes. The enzyme mix is based on a genetically engineered RT polymerase with enhanced thermal stability resulting in an increased specificity, higher cDNA yield and an improved efficiency for highly structured and long cDNA fragments.
The lyophilisate contains all reagents required for RTPCR (except template and primer) in one tube to ensure fast and easy preparation with a minimum of pipetting steps. The premium quality enzyme mix, ultrapure dNTPs and the optimized complete reaction buffer ensure superior amplification results.
SCRIPT RT-qPCR ProbesMaster is used to amplify double-stranded DNA from single-stranded RNA templates. In the RT step the reverse transcriptase synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template. In the first cycle of the PCR step Taq DNA polymerase synthesizes DNA molecules complementary to the cDNA, thus generating a double-stranded DNA template. During subsequent rounds of cycling the DNA polymerase exponentially amplifies this double-stranded DNA template.
In one-step RT-qPCR all components of RT and PCR are mixed in one tube prior to starting the reaction and thus carried out sequentially without opening the tube.
This offers tremendous convenience when applied to analysis of single targets from multiple samples of RNA and minimizes the risk of contaminations.
SCRIPT RT-qPCR ProbesMaster Lyophilisate
Preloaded lyophilisates containing SCRIPT Reverse Transcriptase, Hot Start Polymerase Ab+, dNTPs, Reaction Buffer, MgCl2 and stabilizers.
PCR grade water
SCRIPT RT-qPCR ProbesMaster Lyophilisate is delivered in PCR reaction tube strips or 96-well plates preloaded with a complete qPCR master mix in a dry, room temperature stable format. The lyophilisate combines highest performance with convenience of use and stability. There is no need for freezing, thawing or pipetting on ice. The few remaining pipetting steps minimize the risk of errors or contaminations.
Each vial contains all components (except primers and template) required for a 20 μl RT-qPCR assay.
To perform the assay, only fill up the vials with a mix of primers and RNA template.
Targets can generally be detected from 10 pg to 500 ng polyA RNA or 10 pg to 1 μg total RNA. Even lower amounts of RNA may be successfully amplified by using highly expressed transcripts.
RT-qPCR assay preparation
1. Preparation of the RNA/Primer Mix
Add the following components to a nuclease-free microtube and mix by pipetting gently up and down:
|Component||stock conc.||final conc.||1 assay|
|RNA template||10 pg - 1 μg|
|forward Primer||10 μM||400 - 600 nM||0.8 - 1.2 μl|
|reverse Primer||10 μM||400 - 600 nM||0.8 - 1.2 μl|
|RNase-free water||fill up to 20 μl|
Incubate the mixture at 70°C for 5 min and place it at room temperature for 5 min.
3. Dispensing the master mix
Dispense 20 μl of the RNA/Primer Mix to each lyophilisate containing tube or well of the plate.
Reverse transcription and thermal cycling:
Place the vials in a PCR cycler and start the following program.
|50-55 °C||10-15 min||1x|
|60-65 °C 3)||1 min 4)||35-45x|
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary. Note that optimal reaction times and temperatures should be adjusted for each particular RNA / primer pair.
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