我们热爱生命科学!-生物行
当前位置: 主页 > English > Technology > Methods > RNA

RNA synthesis from plasmid-borne sequences

时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击: 449次
Tuesday, November 18, 2003

Description
In vitro RNA synthesis from plasmid-borne sequences under the control of T phage promoters


Procedure
1) Linearize the plasmid at, or near, the terminus of the insert cDNA with a suitable restriction endonuclease.

2) Extract plasmid DNA with an equal volume of phenol/chloroform and an equal volume of chloroform prior to ethanol precipitation.

3) Linearized plasmid DNA is resuspended in DEPC-treated, autoclaved TE, pH7.5 at a concentration of approximately 200ng/ul prior to use.

4) The standard reaction conditions are set up containing the following quantities:

2ul (400ng) linearized plasmid DNA
20U ribonuclease inhibitor
2ul 100mM DTT
4ul 5 x T3/T7 RNA polymerase buffer
4ul 2.5mM NTPs
Sterile, DEPC treated H2O to a final volume of 19.5ul
25U (0.5ul) T3/T7 RNA polymerase

The reaction mixture is incubated at 37C for 60 minutes.

5) DNA template is removed by the addition of 25U RNase-free DNase I and a further incubation for 30 minutes at 37C. The reaction mixture is phenol/chloroform extracted twice, ethanol precipitated, vacuum dried and resuspended in 25ul sterile, DEPC-treated TE, pH7.5.

6) RNA can be analysed be electrophoresis using denaturing conditions in an agarose/formaldehyde gel system.



Recipes
100mM DTT (Gibco-BRL)
Ribonuclease inhibitor (RNasin, Pharmacia)
T7 or T3 RNA Polymerase (Gibco-BRL)
2.5mM rNTPs (Promega)
5 x T7/T3 RNA Polymerase Buffer (200mM Tris.Cl pH 8.0, 125mM NaCl, 40mM MgCl2, 10mM spermidine, Gibco-BRL)
RNase-free DNase I (Pharmacia)
DEPC-treated, autoclaved, nano-pure water



Supplies


Tips

(责任编辑:泉水)
顶一下
(0)
0%
踩一下
(0)
0%
------分隔线----------------------------
发表评论
请自觉遵守互联网相关的政策法规,严禁发布色情、暴力、反动的言论。
评价:
表情:
用户名: 验证码:点击我更换图片