• 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

        Home-made Taq Polymerase Purification

        互联网

        1610

         

        Home-made Taq Polymerase Purification

        I also have the bacteria containing the clone. It appears to produce lots of Taq and is quite stable.
        The proceedure takes 4 days start to (15 000 units of Taq) finish.
        The Taq also appears very stable and reliable. I made 15 mls a year ago and it still works fine.

        Reference: Engelke, D. R. et al. Anal. Biochem. 191:396-400 (1990).
        Pluthero, F. G. et al. NAR. 21:4850-4851 (1993).

        Day 1

        1) Inoculate two 2L flasks of TB/amp (500ml) with 15ml of an overnight of Taq bugs. These volumes may be scaled down.

        2) Grow to an OD600 of 0.6 (approx mid log)

        3) Add IPTG to 0.5mM (0.119gm/litre), grow o/n but not for more than 16hrs.

        Day 2

        N.B: All the following proceedures should be carried out on ice or at least 4°C.

        4) Collect cells by centrifugation (3.5K / 15 mins / 4°C) and resuspend in 40ml buffer A.

        5) Add an equal volume of buffer B (45-50ml) and incubate at 75°C for 1hr, with periodic mixing.

        6) Centrifuge ( 8K / 15mins / 4°C).

        7) Add 1.86mg of KCl / ml of supernatant.

        8) Aliquot equal volumes of supernatant into each of 2 x 250ml centrifuge tubes (preferably conical) containing 75ml of washed Sigma DP-1 cation exchange resin (packed volume). This should be washed 2 x with sterile water and 4 x with ice cold Buffer B.

        9) Vortex tubes well and incubate on a shaking platform (30mins / 4°C).

        10) Centrifuge (approx 3K / 2min / 4°C) to pellet resin and discard supernatant.

        11) Wash resin 4 x with 100-200ml of ice cold buffer B, remove supernatant by aspiration.

        12) Elute 3 x with one packed bed volume of ice cold buffer C.

        13) Add 30gm (NH4 )2 SO4 / 100ml of eluate while stirring rapidly.

        N.B: At this point it is of great advantage if you use conical or round bottomed tubes. i.e. 50ml tubes for the 8x50 rotor. Prior to this the sample may be handled in 250ml centrifuge bottles for ease of use.

        14) Centrifuge at 12-15Krpm for 10mins at 4°C.

        15) Resuspend pellet (weakly translucent) in 25-35ml of buffer C.

        16) Dialise 2 x against 2L of dialysis buffer (6-18hrs / 4°C).

        Day 3

        17) Titre by assaying serial dilutions cf comercial Taq.

        18) Aliquot concentrated Taq polymerase and store at -20°C.

        Reagents

        Terrific broth (TB); per litre
        12gm tryptone
        24gm yeast extract
        4ml glycerol (autoclaved).
        100ml 0.17M KH2 PO4 (2.31gm/100ml) / 0.72M K2 HPO4 (12.54gm/100ml); Autoclaved separately.

        Buffer A (Require 100ml)
        50 mM Tris (pH 7.9)
        1mM EDTA
        50mM Dextrose

        Buffer B / 100ml (Require 1000ml)
        20mM Hepes (pH7.9) 2ml (1M)
        1mM EDTA 1ml (0.1M)
        0.5% Tween-20 0.5ml
        0.5%NP-40 0.5ml
        0.5mM PMSF
        50mM KCl

        Buffer C / 500ml (Require 500ml)
        20mM Hepes (pH 7.9) 10ml (1M)
        1mM EDTA 5ml (0.1M)
        0.5% Tween-20 2.5ml
        0.5%NP-40 2.5ml
        0.5mM PMSF
        200mM KCl

        Dialysis Buffer / 2L (Require 2000ml)
        20mM Hepes (pH 7.9) 40ml (1M)
        1mM EDTA 4ml (0.5M)
        0.5mM PMSF
        100mM KCl
        50% glycerol 1L
        1mM DTT

        Dilution Buffer Required to dilute Taq
        20mM HEPES (pH 7.9)
        0.1mM EDTA
        100mM KCl
        50% glycerol

         

        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