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Realtime PCR Master Mix Containning All Compositions Except Primers and Probes

Realtime PCR Master Mix SYBR Green Realtime PCR Master Mix

This product is the master mix for QPCR by fluorescence detection based on Taq DNA polymerase. Product generated with Toyobo’s ReverTra Ace -α- cDNA synthesis kit can be used directly in probe assays and intercalator assays just by 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 PRISM 7700 etc. We have confirmed that passive reference does not affect 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, resulting in 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 TaqMan Assay and Hybridization Probe Assay with the preparation of separate primers and probes. SYBR Green Realtime PCR Master Mix is a solution containing Realtime PCR Master Mix and SYBR 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 TaqMan Assay (using Realtime PCR Master Mix).

1. Intercalator Assay with SYBR Green Realtime PCR Master Mix1st strand cDNA synthesis was performed with HeLa total RNA (100 ng) as the template, using ReverTra Ace -α- (Code No. FSK-101) and random primer, and then samples prepared using a fivefold dilution series starting at ten-fold dilution (1-7). PCR (amplification area: 295 bp) was performed with β-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. TaqMan Assay with Realtime PCR Master Mix
The same samples obtained from the seven step dilution series in example 1 were used to perform TaqMan assay with Pre-Developed TaqMan Assay Reagents β-actin (Applied Biosystems) (The sample was 1/10 the volume of the PCR reaction mix). PCR was performed using ABI PRISM7700, 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 PRISM 7700 among others.
  • Suitable for a Broad Range of Assay Systems
    Realtime PCR Master Mix is suitable for probe assays including TaqMan Assay and Hybridization Probe Assay. SYBR Green Realtime PCR Master Mix already contains SYBR Green I and is thus suitable for QPCR utilizing a fluorescent intercalator displacement assay with 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.

One-Point Advice

  • Storage
    As a general rule, store at -20°C away from direct sunlight. However, it can be stored at 4°C for periods of three weeks when used continuously. In this case, it should be stored away from direct sunlight. We recommend storage in a light resistant container or the aluminum bag supplied.
  • Primer Set Up
    In general, short primer sequences are desirable for amplification as they help to retain sensitivity and amplification efficiency and inhibit nonspecific reactions. Primers should be designed to yield a product less than 200 bp in size. Also, it is desirable to avoid GC clusters etc. (especially at 3' end) because they may cause nonspecific reactions such as the formation of primer dimer. Care should be taken to prevent nonspecific reactions, especially in intercalator assays.
  • Use of 1st Strand cDNA
    The direct use of first strand cDNA synthesis reaction mix may inhibit PCR due to the reverse transcriptase activity even if it has already been heat-treated. We recommend dilution with sterile water prior to us