Realtime PCR Master Mix

Realtime QPCR using TaqMan##R### assay, hybridization probe assay and other methods.This product is the master mix for QPCR by fluorescence detection based on Taq DNA polymerase. Product generated with Toyobo's ReverTra Ace -(alpha)-R cDNA synthesis kit can be used directly in probe assays and intercalator assays justby dilution. It contains BSA, and is suitable for glass capillary systems such as LightCycler. In addition, a passive reference dye is also included making it suitable for use with ABI PRISMR 7700 etc. We have confirmed that passive reference does notaffect non-utilizing systems such as LightCycler. This master mix contains anti-Taq high anti-Taq monoclonal antibody, so polymerase activity is inhibited at room temperature prior to PCR allowing Hot Start PCR. This reduces extra bands, resultingin high specificity PCR. Realtime PCR Master Mix is a double-concentrated reaction mix containing Hot Start Taq DNA polymerase, and the reagents and passive reference, etc. required to perform PCR. It can be made suitable for probe assays including TaqManR Assay and Hybridization Probe Assay with the preparation of separate primers and probes. SYBRR Green Realtime PCR Master Mix is a solution containing Realtime PCR Master Mix and SYBRR Green I, and can be made suitable for intercalator assays with the preparation of a separate primer.

Application: Quantitative Determination of Gene Expression by Two-Step RT-PCR

Allows quantitative determination of RNA using cDNA as the sample.

SNP Typing

Allows SNP typing etc. with TaqManR Assay (using Realtime PCR Master Mix).

1. Intercalator Assay with SYBRR Green Realtime PCR Master Mix

1st strand cDNA synthesis was performed with HeLa total RNA (100 ng) as the template, using ReverTra Ace -(alpha)-R (Code No. TYB-FSK-101) and random primer, andthen samples prepared using a fivefold dilution series starting at ten-fold dilution (1-7). PCR (amplification area: 295 bp) was performed with (BETA) -actin as the target using the sample prepared in the above procedure (the volume of the sample was 1/10 of the PCR reaction mix). PCR was performed using LightCycler, at 95°C for 30 seconds, followed by 40 cycles of 95°C for 0 second, 60°C for 0 second, and 72°C for 30 seconds. As a result, excellent amplification curves were obtained for all concentrations (The non-amplification curve for sterile water is blank).

2. TaqManR Assay with Realtime PCR Master Mix

The same samples obtained from the seven step dilution series in example 1 were used to perform TaqManR assay with Pre-Developed TaqManR Assay Reagents (BETA) - actin (Applied Biosystems) (The sample was 1/10 the volume of the PCR reaction mix). PCR was performed using ABI PRISMR7700, at 95°C for 1 minute, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. As a result, excellent amplification curves were obtained for all concentrations (The non-amplification curve for sterile water is blank).

Feature and Advantages: Easy to Use

Double-concentrated PCR master mix is ready to use after addition of sample, primer, probe and sterile water to make twice the volume of the mix.

Broad Versatility

Suitable for Roche Diagnostics' LightCycler and Applied Biosystems' ABI PRISMR 7700 among others.

Suitable for a Broad Range of Assay Systems Realtime PCR Master Mix is suitable for probe assays including TaqManR Assay and Hybridization Probe Assay. SYBRR Green Realtime PCR Master Mix already contains SYBRR Green I and is thus suitable for QPCR utilizing a fluorescent intercalator displacementassaywith unlabeled primers.

Available for Hot Start PCR

Realtime PCR Master Mix contains anti-Taq monoclonal antibody, so Hot Start PCR is possible without any extra processing.