• 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

        Alcohol Precipitation of DNA

        互联网

        1143

         

        The major considerations in using alcohol to precipitate DNA are:

        Temperature: -20°C is optimal, but 0°C can be used for >20 ng/mL

        2) Amount: For small amount of DNA (<100 ng, i.e. too little to reliably see a pellet) the use of glycogen (1 µL of a 20 mg/mL stock, (Roche 10901393001 ) will increase yield and allow visualization of the pellet.

        3) Time: Optimal precipitation requires >1 hr at -20°C.

        4) Speed of centrifuge :  High speeds are important for small amounts or small oligos.  Typically they should be spun at >12,000 rpm (> 12,000 g) in a microcentrifuge.

        5) Mg ++ [10 uM] helps pellet oligonucleotides (<100 bp).  Otherwise Mg should be avoided because it may activate DNAses.

        6) Avoid co-precipitates: EDTA (>10 mM) and Ca (>1 mM) will precipitate at the concentrations indicated in alcohol solutions. SDS will also precipitate when a salt other than NaCl is employed.

        7) Choice of alcohol: Isopropanol has the advantage of requiring less volume.  Typically 0.6 vol to 1 vol. of isopropanol is added to 1 volume of DNA/salt soulution.  The higher amount is useful for smaller RNA fragments.  In contrast, 2 volumes of ethanol is typical.)  However, isopropanol has the disadvantage of coprecipitating more salts and is less volatile (so it is slow to air dry) compared to ethanol.

        8) Choice of salt:

        NaOAc , pH5.2, [0.3M]: This is the standard. (10x Stock concentration = 3 M)

        NaCl [0.2M]:  Useful if SDS is included because it is more soluable in NaCl (Stock conentrations vary 1M - 5M)

        NH4 OAc [2 - 2.5M]: Less coprecipitation of dNTPs, good for purifying oligos. However, NH4 + ions inhibit polynucleotide kinase).

        LiCl [0.8M]: Soluble in a higher concentration of ethanol which is useful for the precipitation of RNA. However Cl- inhibits the initiation of protein synthesis and RNA will display varying solubility based on size.

         

        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