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SCRIPT Transcriptase Reversa | Síntese de cDNA Cellco
SCRIPT Transcriptase Reversa
Ficha técnica
| Código | SCRIPT Transcriptase Reversa |
| Categoria | Síntese de cDNA |
| Marca | Cellco |
Descrição Geral
SCRIPT Transcriptase Reversa | Síntese de cDNA - Cellco
Transcriptase reversa de alta sensitividade e especificidade
For in vitro use only!
Envio: Shipped on blue ice
Condições de armazenamento: Store at -20 °C
Avoid freeze/thaw cycles.
Validade: 12 months
Purity: free of endo- and exodeoxyribonucleases, phosphatases and ribonuclease
Form: liquid
Concentration: 200 units/µl
Form: liquid
Concentration: 200 units/µl
Kit contents:
SCRIPT 3.0 Reverse Transcriptase (blue cap)
200 units/µl in 20 mM Tris-HCl , 5 mM DTT, 50% pH 7.5 (25°C) glycerol (v/v) and stabilizers.
200 units/µl in 20 mM Tris-HCl , 5 mM DTT, 50% pH 7.5 (25°C) glycerol (v/v) and stabilizers.
5x SCRIPT 3.0 RT Buffer complete (red cap)
Tris-HCl (pH 8.3), KCl, MgCl2 and DTT.
Tris-HCl (pH 8.3), KCl, MgCl2 and DTT.
DTT stock solution (purple cap)
100 mM DTT
100 mM DTT
Applications:
Extremely sensitive and highly specific RT-PCR, synthesis of highly structured cDNA fragments, DNA labelling.
Extremely sensitive and highly specific RT-PCR, synthesis of highly structured cDNA fragments, DNA labelling.
Descrição:
SCRIPT Reverse Transcriptase is a genetically engineered version of M-MLV Reverse Transcriptase (M-MLV RT) with reduced Rnase H activity and increased thermal stability. The enzyme is a RNAdirected DNA polymerase that synthesizes a complementary DNA strand initiating from a primer using single-stranded RNA. Its enhanced thermal stability in combination with the deactivated RNase H activity results in an increased specificity, higher cDNA yield and an improved efficiency for full length cDNA synthesis compared with standard M-MLV RT.
Recommended protocols for cDNA synthesis:
Sample denaturation is particularly recommended for RNA targets that exhibit a high degree of secondary structure, for self- or cross-complementary primers and for initial experiments with new targets. For many standard combinations of RNA and primers heat treatment may be omitted with no negative effect on results.
1a Assay set-up without sample denaturation (standard RNA/primer combinations)
Assay preparation
Add the following components to a nuclease-free microtube. Pipett on ice and mix the components by pipetting gently up and down. In general, water, RNA and primers should be mixed together before the remaining components are added.
| Component | Stock conc. | Final conc. | 20 µl assay |
| SCRIPT 3.0 RT buffer complete | 5x | 1x | 4 µl |
| RNA template1 | - | x µl | |
| Primer Fw and Rv2 | 10 µM | 1 - 5 µl | |
| SCRIPT 3.0 RT | 200 units/ µl | 100 units | 0,5 µl |
| RNAse-free water | - | - | up to 20 µl |
| DTT | 100 mM | 5 mM | 1 µl |
| RNAse Inhibitor3 (optional) | 40 units / | 40 units | 1 µl |
| dNTP mix | 10 mM | 500 µM | 1 µl |
1) Total RNA: 10 pg - 5 µg or mRNA: 10 pg - 500 ng
2) Gene-specific primer: 10-20 pmol (50-100 ng) or Oligo-dT 15-25 primer: 50 pmol (300 ng) or Random-primer: 50 pmol (100 ng)
3) Addition of 20-40 units of RNAse Inhibitor per assay is recommended and may be essential when working with low amounts of starting RNA. NOT PROVIDED in this kit.
NOTE:
Adding of up to 5 mM DTT may increase the yield and is recommended for individual optimization
100 units (0,5 µl) of enzyme is recommended for standard assays but increasing the amount of enzyme to 200 units (1 µl) per assay may show even higher transcription yields under selected assay conditions.
1b. Assay set-upRT-PCR assay with sample denaturation: (RNA/primer with a high degree of secondary structure)
NOTE: Sample denaturation is particularly recommended for RNA targets that exhibit a high degree of secondary structure, for self- or cross-complementary primers and for initial experiments with new targets. For many standard combinations of RNA and primers heat treatment may be omitted with no negative effect on results.
Preparation of the RNA Template / Primer Mix
Add the following components to a nuclease-free microtube and mix by pipetting gently up and down.
| Component | [ Stock ] | [ Final ] | [ Final ] |
| RNA template1 | - | 10 pg - 5 µg | x µL |
| Primer forward2 | 10 µM | 10 - 50 pmol | 1 - 5 µL |
| Primer reverse2 | 10 µM | 10 - 50 pmol | 1 - 2 µl |
| RNAse-free water | - | - | up to 10 µl |
1) Total RNA: 10 pg - 5 µg or mRNA: 10 pg - 500 ng
2) Gene specific primer: 10-20 pmol (50-100 ng) - Oligo-dT15-25 primer: 50 pmol ( 300 ng) - Random primer: 50 pmol (100 ng)
Denaturation and primer annealing
Incubate the mixture at 70 °C for 5 min and place it at room temperature for 5 min if using specific primer or on ice if using Oligo-dT or Random primer.
Preparation of the RT-PCR Mix
Add the following components to the RNA template and primer mix and mix by pipetting gently up and down.
| Component | Stock conc. | Final conc. | 20 µl assay |
| SCRIPT RT buffer complete | 5x | 1x | 4 µl |
| DTT stock solution | 100 mM | 5 mM | 1 µl |
| RNAse Inhibitor | 40 units / µl | 40 units | 1 µl |
| dNTP mix | 10 mM each | 500 µM each | 1 µl |
| SCRIPT RT Enzyme | 200 units/µl | 100 units | 0,5 µL |
NOTE:
- Adding of up to 5 mM DTT may increase the yield and is recommended for individual optimization.
- Addition of 20-40 units RNase inhibitor per assay is recommended and may be essential when working with low amounts of starting RNA. NOT PROVIDED in this kit.
- 100 units (0,5 µl) of enzyme is recommended for standard assays but increasing the amount of enzyme to 200 units (1 µl) per assay may show even higher transcription yields under selected assay conditions.
2. First-strand cDNA synthesis:
- Place the vials in a PCR cycler and start the following program.
- Incubate the reaction mix at 55 ºC for 30-60 min if using gene-specific primers. If using Oligo-dT or Random primers incubate at 42 ºC for 10 min followed by incubation at 55 ºC for 30-60 min.
| Step | Temp. | Time |
| Reverse transcription1 | 55 ºC | 30-60 min |
| Inactivation of RT2 | 70 ºC | 10 min |
1) The optimal time depends on the length of cDNA. Incubation of 60 min is recommended for cDNA fragments of more than 2,000 bp length. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 60 °C for difficult templates with high secondary structure.
2) Optional to heat inactivate the Reverse Transcriptase.
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.
3. RNA removal (optional)
Add 2 units DNase-free RNase and incubate at 37 °C for 20 min. The cDNA can now be used as template in PCR or be stored at -20 °C. Apply 2-5 µl of the RT assay for further amplification in PCR.
However, some specific DNA applications may require the prior inactivation of the remaining RTase or the enzymatic removal of RNA.
Activity:
Activity and stability tested in first strand cDNA synthesis.
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