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Ribonuclease T2, Recombinant - Assay
Source:
Aspergillus oryzae
CAS:
37278-25-4
EC:
4.6.1.19
Ribonuclease T2, Recombinant
Method
This method used at Worthington Biochemical Corporation is based off of Uchida, T. (1966) looking at the release of acid soluble oglionucleotides following the digestion of yeast RNA.
Unit Definition: One unit will cause an increase in absorbance of 1.0 at 260nm when yeast RNA is hydrolyzed at 37°C, pH 4.5 in 15 minutes.
Reagents
200mM Sodium Acetate Buffer, pH 4.5: Measure 350ml deionized water into an appropriate beaker with stirbar, and warm to 37°C using heated stirplate. While heating, add 13.6g sodium acetate, trihydrate (FW 136.08). Adjust pH to 4.5 with 5N HCl and final volume to 500ml with deionized water. Solution may be stored up to one year in 4°C cold room.
20mM EDTA, pH 8.0: Dissolve 745mg EDTA in 80ml reagent grade water. Adjust pH to 8.0 with dilute NaOH and bring to a final volume of 100ml.
17.7mM Uranyl Acetate in 25% (v/v) Perchloric Acid solution: Add 35.6ml of stock 70% Perchloric Acid Solution to 64.4ml of deionized water. Then add 751mg Uranyl Acetate. Solution may be stored up to one year in 4°C cold room covered with foil.
RNA Solution: Prepare a fresh solution of 12 mg/ml RNA (Roche Catalog #109223 – consult supervisor if this material is unavailable) by suspending in approximately 80% of final volume of reagent grade water. Slowly add dilute NaOH until pH reaches 8.0. Note: the pH may drop as RNA dissolves. Repeat pH adjustments until RNA is completely dissolved and pH remains steady at 8.0 ± 0.1. Adjust final volume with reagent grade water. Check A260 of RNA using a 350X dilution to ensure RNA is at or near 12 mg/ml. The A260 of a 350x dilution should be 0.85 ± 0.05.
Note: (A260) x (Dilution Factor) x (40 μg/ml) = mg/ml RNA.
If concentration of RNA is too high, dilute as needed with deionized water until A260 reads at desired range. If concentration of RNA is too low, discard and prepare a new RNA Solution.
Enzyme
For RT2R, dissolve powder at 10 mg/ml in cold 200 mM sodium acetate buffer, pH 4.5.
Dilute enzyme as necessary using same cold buffer.
Procedure
Buffer mixture: Pipette according to the number of reactions, at the following proportion:
0.30 ml deionized water
0.25 ml 200 mM Sodium Acetate Buffer, pH 4.5 0.10 ml 20 mM EDTA Solution
0.25 ml RNA Solution
For 40 tubes, prepare buffer mixture of: 12 ml deionized water
10 ml 200 mM Sodium Acetate Buffer, pH 4.5 4 ml 20 mM EDTA Solution
10 ml RNA Solution
Include at least 3 tubes to serve as blanks. Incubate tubes in a 37°C water bath for 5 minutes. At timed intervals, add 0.1ml diluted enzyme (0.1ml buffer for blanks). Immediately mix by swirling incubate exactly 15 minutes at 37°C. At 15 minutes time, stop reaction by adding 0.25ml uranyl acetate/perchloric acid solution and remove tube from bath. Mix by swirling and allow to stand at room temperature for 15minutes. Centrifuge tubes in bench-top centrifuge at 2500 rpm for 10 minutes. Withdraw 0.200ml clear supernatant and add to tubes containing 4.80ml of deionized water. Mix and read A260’s versus deionized water.
Calculation
Avg Blank: Average blank A260 values together.
5 = Total volume (ml) of assay (0.200ml stopped reaction + 4.8ml DI water)
1.25 = Volume (ml) of stopped reaction
0.1 = Volume (ml) of enzyme used
0.2 = Volume (ml) reaction mix used in assay
1.25 = Volume (ml) of stopped reaction
0.1 = Volume (ml) of enzyme used
0.2 = Volume (ml) reaction mix used in assay
Ribonuclease T2, Recombinant Products
Description
Activity
Code
Cat. #
Size
Price
Description
Ribonuclease T2, Recombinant, Animal Free/AF
Source:
Komagataella phaffii (previously P. pastoris)
Recombinant Aspergillus oryzae (non-mammalian) broad specificity Ribonuclease T2 produced in Komagataella phaffii. Chromatographically purified, Animal Free/AF. Supplied as lyophilized powder.
Store at 2-8°C.
Code
RT2R
Product details
LS01501
50 ku
$90.00
LS01502
250 ku
$355.00
LS01505
Bulk
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