Reverse Transcriptase, Recombinant HIV Assay

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Reverse Transcriptase, Recombinant HIV Assay

Method

Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) _12-18 as the template-primer in 20 minutes at 37° C.

Reagents

0.05 M Tris, pH 8.3, containing 0.008 M MgCl₂
1 mg/ml polyadenylic acid in water (poly A)
DNA primer:Oligo d(T)_12-18
1 μ mole dTTP/mL stock solution
[methyl-³H]-Thymidine 5'-triphosphate (³H-dTTP)
dTTP-³H-dTTP working mix: Add 1-2 μL ³H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 10⁵ cpm/mL
1% bovine serum albumin
10% perchloric acid
1% perchloric acid
Buffer substrate reaction mixture: Prepare fresh, immediately before use:
For each 1mL of reaction mixture required mix:
0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl₂
0.3 mL 1 mg/mL poly(A) RNA template
0.005 mL 0.02 mg/mL oligo d(T)_12-18 DNA primer
0.02mL 1% BSA

Enzyme

dilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl₂ containing 0.1 mg/mL (1%) BSA

Procedure

Pipette into each tube as follows:

Buffer substrate mix 0.1 mL
dTTP-³H³-dTTP 0.1 mL
Enzyme 5-10 μL

Incubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2μ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.

Calculation

\frac{\text{Units}}{\text{ml}}\text{ = } \frac{\text{reaction cpm x 10}}{\text{total cpm x reaction volume (ml)}}

\frac{\text{Units}}{\text{mgP}}\text{ = } \frac{\frac{\text{Units}}{\text{ml}}}{\frac{\text{mgP}}{\text{ml}}\text{ (Lowry)}}

Reverse Transcriptase, Recombinant HIV Products

Description

Reverse Transcriptase, Recombinant, HIV

Source:

Recombinant protein produced in E. coli

Chromatographically purified dimeric form with M.W. of 66 kDa and 51 kDa. A solution in 10mM potassium phosphate, pH 7.4, 1mM DTT and 20% glycerol.

Store at -20°C.

…

Dry Ice required

Activity

≥5,000 units per mg protein

Certificate of Analysis

Code

RTHIV

Product details

LS05003

200 un

$95.00

LS05006

5x200 un

$375.00

LS05000

Bulk

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Buffer substrate mix	0.1 mL
dTTP-³H³-dTTP	0.1 mL
Enzyme	5-10 μL

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Reverse Transcriptase, Recombinant HIV - Assay

Reverse Transcriptase, Recombinant HIV Assay

Reverse Transcriptase, Recombinant HIV Products