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SunScript™ One Step RT-PCR Kit

SunScript™ One Step RT-PCR Kit
SunScript™ One Step RT-PCR Kit is an easy and reliable system for fast, specific and sensitive end point RT-PCR reactions. The kit contains all components needed for both reverse transcription and PCR amplification in a single tube “one step” reaction that minimizes contaminations and facilitates multiple sample processing.
 
The system uses an enzyme mix containing SunScript Reverse Transcriptase RNaseH-, a high quality HotStart Taq Polymerase and RNase inhibitor for a robust and effective amplification with starting total RNA amounts of 1μg to picograms. SunScript RNaseH- is an engineered version of the well characterized HIV-1 RT with increased thermostability and absence of RNaseH activity. It minimizes RNA degradation for higher cDNA yields and makes it posible to perform the reverse transcription step at temperatures up to 85ºC if needed.*
 
SunScript™ One Step RT-PCR Kit is optimized for amplifying difficult RNAs with high degree of secondary structure or high GC content. The reverse transcription reaction can be effectively achieved while the Taq Polymerase remains inactive due to blocking antibodies. The inactivation/activation step at 95ºC provides an automatic “hot start” for the Taq Polymerase to amplify the reverse transcribed cDNA.
 
The 2x Reaction Buffer contains a proprietary formulation which has been optimized for both the reverse transcription and the PCR reaction including dNTPs, MgCl2 and stabilizers (to provide a final concentration of 250 nM for each dNTP and 3.5 nM MgCl2 in the reaction).
 
* The recommended temperature for the RT reaction is 60ºC for most samples
 

Advantages

  • Fast, specific and sensitive system for end point RT-PCR reactions
  • Performs both reverse transcription and PCR in the same single tube
  • Allows “one step reaction” to minimize contaminations
  • Convenient system to process multiple samples
  • SunScript technology enables amplification of difficult RNAs
  • High reproducibility of reactions
  • Robust and reliable

Reaction Temperatures Up To 85ºC

1s1
100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for GAPDH gene (product length 305 bp). The RT reaction can be performed at temperatures up to 85ºC, making this the best choice for detecting difficult RNAs, with high degree of secondary structure.

Detects Genes With Different Expression Levels

1s2
100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for the indicated genes (GAPDH, HERC, GUSB, CLTC and ACTIN). The kit easily detects genes with vastly different expression levels.

Sensitivity From Micrograms to Picograms Of Starting RNA

1s3
The indicated amounts of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for GAPDH cDNA (product length 305 bp). The sensitivity of the kit is good enough to detect starting RNA amounts in the picogram range.

Highly Reproducible Results

1s4
100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and specific primers for the indicated genes (4 replicates per gene). Replicates show similar sensitivity and yield, making this kit the best choice for processing multiple samples with minimum deviations.

Amplification Of Difficult RNAs

1s5
100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript™ One Step RT-PCR Kit and four different specific primer pairs for a fragment of the TAFII100 cDNA with 75% GC content (product lengths 430-470 bp). The RT-PCR protocol with sample denaturation was performed here and the cDNA synthesis step was performed at 60ºC. The four oligo pairs produced effective amplification bands despite the elevated GC content of the template RNA.

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