• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        【分享】RT-PCR Protocol (Reverse Transcriptase-PCR)

        丁香园论坛

        1921
        Reverse Tranase-PCRThis method was submitted by Jim Hutchins from the University of Mississippi Medical Center.

        The following is a great RT-PCR protocol developed by Ignacio Rodriguez at the National Eye Institue, NIH.

        References

        Raineri, I., Moroni, C. and Senn, H.P. (1991).
        Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Res. 19 : 4010-20
        Rodriguez, I.R., Mazuruk, K.,Schoen, T.J. and Chader, J.G. (1994). Structural analysis of the human hydroxyindole- O-methyltransferase gene : presence of two distinct promoters. J. Biol. Chem. 269, 31969- 31977.

        Rodriguez, I.R. and Chader, G.J., A novel method for the isolation of tissue-specific genes, Nucleic Acids Res., 20 (1992) 3528.

        Schoen, T. J., Mazuruk, K., Chader G. J., and Rodriguez, I. R. Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive technique. Biochem. Biophys. Res. Commun., 213 (1995) 181-8.

        Protocol
        PROCEDURE FOR cDNA SYNTHESIS ON DYNABEADS oligo(dT)25 (5/4/94)
        1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1M KCl.

        2. a) Place 100 ul Dynabeads oligo(dT)25 (5 mg/ml) in a 0.5 ml tube.
        b) Bind beads.
        c) Remove liquid.
        d) Add 100 ul TE/1M KCl.
        e) Wash.
        f) Bind beads.
        g) Remove liquid.

        3.a) Add RNA to beads.
        b) Heat to 70 C for 2 min and cool slowly to RT for 10 min.
        c) Bind beads.
        d) Remove liquid.

        4. Resuspend beads in :
        2.5 ul Buffer undefined (200 mM Tris-HCl, pH 8.3,1.0 M KCl)
        2.5 ul Buffer undefined (30 mM MgCl2 and 15 mM MnSO4)
        20.0 ul dNTPs (2.5 mM each)
        1.0 ul 32P-dCTP (5 uCi)
        1.0 ul RNasin-Pharmacia
        2.0 ul Super II RT (200 U/ul)(Gibco BRL # 18064-014)
        5.0 ul Retrotherm RT (1 U/ul) (Epicentre Technologies # R19250)
        16.0 ul H20
        -----------------
        50 ul

        ~undefined These buffers are supplied with the Retrotherm RT.

        5. Remove 1 ul of reaction. This represents total 32P counts for use in calculating the amount of cDNA synthesized.

        6. Heat at 40 C for 30 min.

        7. Heat at 70 C for 1 hr.

        8. Bind beads and remove all liquid.

        9. Wash beads with 100 ul TE, bind beads, remove liquid.

        10. Resuspend beads in 100 ul TE. Count 1 ul of beads to calculate the amount of cDNA synthesized. Use 1 ul of beads per PCR.

        I think the real secret here is the removal of secondary structure by the thermostable RT coupled with the immobilized RNA forming a "reusable" pool of template. This works well with total RNA or a poly(A)+ fraction.

        详细步骤请下载PDF文件:RT-PCR
        http://blog.bioon.com/user3/24773/archives/2007/135911.shtml

        常见问题:
        How can I tell if my RT-PCR product in RNA specific? (Invitrogen)
        http://invitrogen.custhelp.com/cgi-bin/invitrogen....
        Added: Tue May 14 2002, Hits: 3148, Reviews: 0 Write review Cached
        How can I treat my RNA sample before RT-PCR to eliminate DNA contamination? (Invitrogen)
        http://invitrogen.custhelp.com/cgi-bin/invitrogen....
        Added: Tue May 14 2002, Hits: 4492, Reviews: 0 Write review Cached
        How much of the first strand reaction should I add to the PCR? (Invitrogen)
        http://invitrogen.custhelp.com/cgi-bin/invitrogen....
        Added: Tue May 14 2002, Hits: 2569, Reviews: 0 Write review Cached
        How should I prime my cDNA? (Invitrogen)
        http://invitrogen.custhelp.com/cgi-bin/invitrogen....
        Added: Tue May 14 2002, Hits: 2825, Reviews: 0 Write review Cached
        RNase inhibitors and RNases (Invitrogen)
        http://invitrogen.custhelp.com/cgi-bin/invitrogen....
        Added: Tue May 14 2002, Hits: 1465, Reviews: 0 Write review Cached
        What is the highest temperature that reverse traases can be used? (Invitrogen)
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序