Cambio - Excellence in Molecular Biology

In vitro Transcription

In vitro Transcription: In Vitro Transcription

MonsterScript™ Reverse Transcriptase

MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase that completely lacks RNase H activity

Epicentre® Biotechnologies

MonsterScript™ Reverse Transcriptase

MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase that completely lacks RNase H activity

Epicentre® Biotechnologies

MonsterScript™ Reverse Transcriptase is a tmagnesium-dependen thermostable reverse transcriptase that completely lacks RNase H activity. The enzyme's lack of RNase H activity contributes to its ability to make longer cDNAs and more complete full-length libraries of first-strand cDNA molecules compared to other reverse transcriptases. EPICENTRE scientists have demonstrated reverse transcription of 15-kb RNA templates using MonsterScript™ Reverse Transcriptase (Fig. 1).

The MonsterScript 1st-Strand cDNA Synthesis Kit includes MonsterScript RT PreMix, which contains optimized concentrations of dNTPs, Mg2 , and betaine.* Betaine reduces pausing and stops by the reverse transcriptase, enabling improved reverse transcription through difficult sequences, such as regions of high GC content. The kit also contains both an oligo(dT)-containing primer and random nonamer primers.

Quality Control

MonsterScript™ Reverse Transcriptase is free of detectable contaminating DNA exo- and endonuclease and RNase activities.



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MonsterScript™ Reverse Transcriptase

MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase that completely lacks RNase H activity

Epicentre® Biotechnologies

Protocols for: MonsterScript™ 1st-Strand cDNA Synthesis Kit & MonstreScript™ Reverse Transcriptase 

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Epicentre’s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

 

MonsterScript™ Reverse Transcriptase Protocol

(catalogue number MSTA5110 / MSTA5124) 

Please note: all protocols off site are the responsibility of the products supplier

 

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

MonsterScript™ Reverse Transcriptase

MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase that completely lacks RNase H activity

Epicentre® Biotechnologies

Applications

  • First-strand cDNA synthesis for subsequent PCR or real-time PCR.
  • RT-PCR validation of gene expression data obtained from microarray experiments.
  • RT-PCR validation and quantification of gene silencing by RNA interference.
  • Preparation of labeled cDNA probes.  

Benefits

  • No RNase H activity, enabling improved synthesis of full-length cDNA even for long mRNA, using random priming.
  • Thermostable, permitting reverse transcription at temperatures >50°C, which reduces RNA secondary structure and improves priming specificity.
  • MonsterScript RT PreMix contains optimized concentrations of dNTPs, Mg2 , and betaine for superior performance and minimal pipetting steps.
  • The kit includes both an oligo(dT) and random nonamer primers for maximal transcript coverage.
  • First-strand cDNA can be made from picogram amounts of total RNA (Fig. 2).
  • The kit includes a potent RNase Inhibitor to protect the integrity of template RNA.

 

MonsterScript Reverse Transcriptase produces full-length cDNA from mRNA up to or greater than 15kb Figure 1. MonsterScript™ Reverse Transcriptase produces full-length cDNA from mRNA up to or greater than 15 kb The ≈15.2-kb human guanine nucleotide exchange factor, p532, mRNA ('HGNEFp532 mRNA') was reverse transcribed using total HeLa cellular RNA and an oligo(dT)-containing primer in a standard MonsterScript reaction. Two µl of the reverse transcription reaction was used to amplify a 1.3-kb region within 68 bases of the 5'-end of the HGNEFp532 mRNA with the FailSafe™ PCR System. 1A. Detection of the PCR product demonstrates full-length reverse transcription of the HGNEFp532 mRNA by MonsterScript Reverse Transcriptase. 1B. Agarose gel analysis of the PCR reaction shows the 1.3-kb amplicon amplified from the extreme 5'-end of the HGNEFp532 mRNA.
MonsterScript Reverse Transcriptase produces full-length cDNA from mRNA up to or greater than 15kb

 

Figure 2. MonsterScript™ Reverse Transcriptase was used to reverse-transcribe six dilutions (8 x 10-6 to 8 x 10-1 pg) of an RNA transcript into cDNA in 20-µl reactions. Ten microliters of each reverse transcription reaction was then used in 50-µl real-time PCRs, in duplicate, using EPICENTRE's TAQurate™ GREEN Real-Time PCR MasterMix. A PCR efficiency of 99.4% and correlation coefficient of 1.000 was achieved.

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200