AMV Reverase Transcriptase
| Catalog # | Pack size | Price, Euro | SHOP |
| 105400 | 200 u | 39,00 | Add to cart |
| 105410 | 1000 u | 156,00 | Add to cart |
Datasheet for: AMV-Reverse Transcriptase
Description
AMV Reverse Transcriptase (AMV RT) catalyzes the polymerization of DNA using template DNA,RNA or RNA:DNA hybrids (1). It requires a primer (DNA primers are more efficient than RNA primers) as well as Mg2+ or Mn2+. The enzyme possesses an intrinsic RNase H activity. Please refer to the Usage Notes, before using this enzyme.
Source: Purified from avian myeloblastosis virus particles.
Applications of AMV RT include:
• First-strand synthesis of cDNA from RNA molecules (2).
• Sequencing of RNA transcripts (3).
Concentration: 5000 – 15.000 units/ml
AMV Reverse Transcriptase 5X Reaction Buffer: 250 mM TrisHCl, (pH 8,3 at 25°C), 250mM KCl, 50 mM MgCl2, 2,5 mM spermidine and 50 mM DTT.
Storage Buffer: AMV Reverse Transcriptase (AMV-RT) is supplied in 200 mM potassium phosphate (pH7.2 at 4°C), 0,2 % Triton X-100, 2 mM DTT and 50% glycerol.
Unit definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1 mmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C. The reaction conditions are: 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 8.75 mM MgCl2, 10 mM DTT, 0,1 mg/ml acetylated BSA, 1 mM radiolabeled dTTP and 0.25 mM poly(A): oligo(dT). See the unit concentration on the Product Information Label.
Storage condition: -20°C
First-Strand Synthesis of cDNA Protocol
1. Mix in the tube:
- 2 µg of the total RNA (or 50-500 ng of polyA RNA)
- 500 ng of primer for each µg of RNA add water up to 8 µl (<11 µl)
2. Incubate the mixture: 5 min at 70°C, then chill for 5min on ice
3. Add into the mixture:
- 5 µl of 5xRT buffer complete (250mM TrisHCl, pH8,3; 500mM KCl, 15mM MgCl2, 50mM DTT)
- 2,5 µl of dNTP mix (10mM of each dNTP; Cat.-No: 110001 and 110002)
- RNAsin 40 units (optional)
- 2,5 µl sodium pyrophosphate, 40mM (prewarmed to 42°C)
- AMV RT 30 units
- H2O (nuclease-free) – up to 25 µl
4. Mix gently: transfer 5 µl of the reaction mixture to another tube containing 2-5 µCi [α-32P]dCTP. Do not add lable to the remaining 20µl reaction. Note: We recommend using [α-32P]dCTP that is less than 1 week old.
5. Incubate the mixture: for 60 min at 42°C for oligo(dT) primers or at 37°C for random hexamer primers.
6. Enzyme inactivation: Place the reactions, labelled and unlabeled, on ice and add 95 µl of 50 mM EDTA to the labelled (tracer) reaction. The reaction volume should now total 100 µl. The Tracer reaction may be used for an incorporation assay and gel analysis.
7. Perform second-strand synthesis: using the unlabeled first-strand reaction see reference (4). No phenol extraction or ethanol precipitation is necessary.
Sequencing of RNA Transcripts
A protocol for sequencing RNA transcripts may be found in reference (3).
Usage Notes:
1. The formulation of AMV Reverse Transcriptase 5x Reaction buffer is not compatible with M-MLV Reverse Transcriptase.
