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HIV-1 RTase (HIV-1 Reverse Transcriptase)

The human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RTase is an enzyme that can catalyze complementary DNA (cDNA) synthesis from an RNA template. Due to its greater thermostability than comparatives of AMV and MMLV, HIV-1 RTase is currently used for RT-LAMP reactions, in combination with Bst DNA polymerase LF.
No. Size Price Qty Status
C15020-200U 200U $340.00 Inquiry
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package & Component :
Cat. Name Amount
C15020-200U HIV-1 Reverse Transcriptase (5 U/μL) 200 U
10X Isothermal Amplification Buffer 1 mL
100 mM MgSO4 0.4 mL

Source:
Escherichia coli

Purity:
>98% as determined by SDS-PAGE (purified by Ni-NTA chromatography).

Unit Definition:
One unit is defined as the amount of the enzyme incorporates 1 nmol of dTTP into acid-insoluble product in 10 minutes at 50°C.

Reaction Condition:
1X Isothermal Amplification Buffer, supplemented with dNTP mix, template and primer, Incubate at 65°C for one-step RT-LAMP.
10X Isothermal Amplification Buffer: 200 mM Tris-HCl (pH 8.8), 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, and 1% Tween 20.

Storage Buffer:
HIV-1 Reverse Transcriptase is supplied in 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol (v/v).

Storage:
Stored at -20°C. Avoid repeated freeze/thaw cycles.

Notes:
After the reaction is complete, Bst DNA Polymerase and HIV-1 RT can be inactivated by incubation at 80°C for 10 minutes.
First strand cDNA synthesis:
1. Place all required reagents on ice and add each of them following the order suggested below.
Component Amount Final concentration
Oligo (dT)12-18 (50 μM) or random primer mix (60 μM) 1 μL -
Total RNA template X μL 1 μg
Nuclease-Free H2O Y μL -
Total reaction volume 13.5 μL -
2. Heat the tube to 65°C for 10 minutes to denature the secondary structure within RNA template. Immediately cool the tube on ice for 1 minute and centrifuge briefly in microcentrifuge. Add the following components to the annealed primer/RNA template, prepare on ice.
Component Amount Final concentration
5X Reverse Transcriptase Reaction Buffer 4 μL 1X
10 mM dNTPs (each) 1 μL 0.5 mM each
RNase Inhibitor 0.5 μL 20 U/rxn
HIV RTase 1 μL 20 U/rxn
Total reaction volume 20 μL 5 U/rxn
3. Incubate at 42-50°C for 1 hour.
4. Inactivate the reaction at 80°C for 10 minutes. The cDNA products should be store at -20°C.
5. Reaction preparations may be scaled up or down proportionately.

RT-LAMP reaction
1. Place all required reagents on ice and add each of them following the order suggested below.
Component Amount Final concentration
10X Isothermal Amplification Buffer 2.5 μL 1X
100 mM MgSO4 1.5 μL 6 mM final concentration, total 8 mM
10 mM dNTP mix 3.5 μL 1.4 mM each
10X FIP/BIP primers 1 μL 1.6 μM
10X F3/B3 primers 1 μL 0.2 μM
10X LoopF/B primers 1 μL 0.8 μM
RNA template X μL -
Nuclease-Free H2O Y μL -
Bst DNA Polymerase
(Large Fraction)
1 μL 8 U/rxn
HIV-1 Reverse Transcriptase 2 μL 10 U/rxn
Total reaction volume 25 μL -
2. Gently mix the reaction thoroughly to achieve uniform distribution.
3. Incubate at 65°C for 30-60 minutes.
4. MgSO4 (2-10 mM), Bst DNA Polymerase (40-320 U/mL), HIV-1 RT and temperature (50-65 °C) can be adjusted for optimal results.
5. Reaction preparations may be scaled up or down proportionately.