Worthington nuclease and Micrococcus related research and determination scheme

AI Meijie Worthington Nuclease 、 Micrococcus background ：

Micrococcus nuclease catalyzes  DNA  and  RNA  Cut to produce  3'- Nucleotides . It has circumscribed and inscribed  5'- Phosphodiesterase activity . The enzyme catalyzes at sites rich in adenylate or uridine acid and deoxyadenylate or thymidine Acid  RNA  and  DNA  Preferential endohydrolysis . The molecular weight of the enzyme is  16,807  Dalton , And depends on calcium . The best  pH  The value is  9.2, But it will change according to the concentration of ionic calcium .

Unit definition ： A unit corresponds to  37°C、pH 8.0、260 nm  Optical density change at  1.0, Use  DNA  As substrate .

Worthington Nuclease 、 Research on Micrococcus ：

Albumin , Nuclease free  (BSANF)

Deoxyribonuclease  I (DP/D/DCLS/D2/DPFF/DPRF)

E•RASE RNase A/T1 Blend (RCT)

Histone  (H, NHL)

Lysozyme  (LY/LYSF)

Nuclease ,S1 ( SINUC/SINUCL)

nucleic acid 、DNA、 E. coli 、λ/ fragment 、RNA

Phosphatase 、 alkalinity  (CAP/BAPF/BAPC/BAPSF/PC)

Phosphodiesterase  I (VPH)

Phosphodiesterase  II (SPH)

protease  K (PROK/PROKS)

Reverse transcriptase , restructuring  HIV (RTHIV)

Ribonuclease  A (R/RAF/RASE/RS/RPDF)

Ribonuclease  T1, No animal origin  (RT1S)

 

AI Meijie Worthington Nuclease 、 Micrococcus Determination ：

Method ： Is basically  Heins As described by others .(1966)  Based on nuclease digestion  DNA  Release of acid soluble oligonucleotides . A unit corresponds to  37°C  and  pH 8.0  Under specified conditions at  260 nm  Situated  1.0  Optical density change .

Reagents ：

0.1 M  Sodium borate ,pH 8.8

0.01 M  calcium chloride

DNA,2.5 mg/ml： take  25 mg Worthington  Calf thymus  DNA  Dissolve in  10 ml 0.01 M NaCl  Prepared in . Let stand overnight at room temperature , Then stir slowly to produce a solution .

7%  Perchloric acid

0.1%  Bovine serum albumin

enzyme ：

With  1 mg/ml  Dissolve in reagent grade water . stay  0.1%  Dilute albumin to  0.001-0.002 mg/ml  Used to determine .

equation

Program ：

Enter the following pipette  5  Each of the tubes numbered ：

0.1 M  Sodium borate ,pH 8.8 0.1  ml

0.01 M CaCl 2 0.05  ml

0.25% DNA 0.1  ml

stay  37°C  Incubate in a water bath  6-7  Minutes to reach temperature equilibrium . stay  0.001-0.0002 mg/ml  Prepare at least four different enzyme dilutions in the range . Every once in a while , take  0.1 ml  Transfer the appropriate diluent into the corresponding test tube , And replace it in the water bath . Add  0.1 ml 0.1%  Albumin . Incubate  30  minute . By adding  0.5 ml 7%  Perchloric acid stops reaction at regular intervals . Place the test tube in an ice bath  10  minute , Then join  2.7 ml  Reagent grade water . Centrifuge at the highest speed on a clinical centrifuge  15  minute . Extract the supernatant and read  A 260 Compare with blank .

Be careful ： For best results , The sample should be diluted so that the final  A 260 Be situated between  0.2-0.9  Between .