丁香实验_LOGO
登录
搜索
    搜索
    提问
    我要登录
    ｜免费注册
    点赞
    收藏
    wx-share
    分享

    BACs, PACs, P1s and Plasmid Isolation Protocol (For low copy number)

    互联网

    759

    实验试剂

     

    1. Sterile Deionized Water (or TE Buffer)

    2. 70% Ethanol

    3. 96%-100% Isopropanol

    实验设备

     

    1. Microcentrifuge capable of at least 18,000 x g

    2. Sterile 1.5 ml Centrifuge Tubes

    3. 10-15 ml Culture Tubes

    实验步骤

     

    1. Isolate a single colony from a freshly streaked selective plate, and inoculate a starter culture of 2-5 ml LB or YT medium containing the appropriate selective antibiotic. Incubate for ~ 20-24 hr at 37E C with vigorous shaking (~ 300 rpm). Use a flask with a volume at least 4 times the volume of the culture.

    2. Pellet 1.5-5 ml bacteria by centrifugation at 13,000 x g for 3 min at room temperature. Decant or aspirate medium and discard.

    3. Resuspend the bacterial pellet by adding 260 ul of Buffer T1/RNase A solution, and vortexing. Complete resuspension of cell pellet is vital for obtaining good yields.

    4. Add 260 ul of Buffer T2 and gently mix by inverting 5-10 times to obtain a clear lysate. Incubate at room temperature for 5 minutes. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. The lysate should appear viscous. Do not allow the lysis reaction to proceed more than 5 min. (Store Buffer T2 tightly capped when not in use)

    5. Add 260 ul of Chilled Buffer T3 and gently mix by inverting 15-20 times until a flocculent white precipitate forms. Incubate on ice for 5 minutes.

    Do not vortex, as this will result in shearing.

    6. Centrifuge at 18,000 x g for 10 minutes at 4o C. Promptly proceed to the next step.

    7. Carefully aspirate and add the clear supernatant to a HiBind DNA Column assembled in a provided 2 ml collection tube. Ensure that the pellet is not disturbed and that no cellular debris has carried over into the column. Centrifuge for 1 min at 13,000 x g at room temperature to completely pass lysate through the HiBind DNA Column.

    8. Discard the HiBind DNA Column and add 2 ul of the supplied linear polyacrylamides to the 2 ml collection tube containing the cleared cell lysate. Add a 0.7 volume of room temperature isopropanol to the samples. ( 546 ul of isopropanol for 780 ul of cell lysate). Mix the sample by vortexing for 15 seconds.

    9. Centrifuge at 18,000 x g for 10 minutes at room temperature to pellet the DNA. Carefully aspirate or decant the supernatant and discard, making sure not to dislodge the DNA pellet.

    10. Wash the DNA pellet with 500 ul of 70% ethanol. Centrifuge the 2 ml collection tube containing pellet (in the same orientation as before) for10 minutes. Carefully aspirate or decant the supernatant, being careful not to dislodge the DNA pellet. Invert the tube containing the DNA pellet on a paper towel for 10-15 min to air dry the DNA pellet. Note: Ensure that no alcohol droplets are visible after air drying, with out overdrying the DNA pellet. Overdrying of the DNA pellet will make redissolving the pellet difficult.

    11. Redissolve the DNA pellet by adding 30-50 ul of Elution Buffer (10 mM Tris-HCl, pH 8.5), and incubating overnight at room temperature

    12. Yield and quality of DNA: Determine the absorbance of an appropriate dilution of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows: 260 DNA concentration = A x 50 x (Dilution Factor) :g/ml 260 280 A ratio of (A ) / (A ) is an indication of nucleic acid purity. Alternatively, yield (as well as quality) can sometimes be best determined by agarose gel/ethidium bromide electrophoresis.

    相关产品推荐

    LGALS1/LGALS1蛋白/14KDA lectin Beta-galactoside-binding lectin L-14-I Galaptin Lactose-binding lectin 1 Lectin galactoside-binding soluble 1 S-Lac lectin 1蛋白/Recombinant Bovine Galectin-1 (LGALS1)重组蛋白

    LGALS1/LGALS1蛋白/14KDA lectin Beta-galactoside-binding lectin L-14-I Galaptin Lactose-binding lectin 1 Lectin galactoside-binding soluble 1 S-Lac lectin 1蛋白/Recombinant Bovine Galectin-1 (LGALS1)重组蛋白

    ￥69

    mRNA纯化试剂盒|高效、灵敏、高性价比| mRNA Isolation Master Kit

    mRNA纯化试剂盒|高效、灵敏、高性价比| mRNA Isolation Master Kit

    ￥2600

    yscM/yscM蛋白/Low calcium response locus protein Q蛋白/Recombinant Yersinia pseudotuberculosis serotype I Yop proteins translocation protein M (yscM)重组蛋白

    yscM/yscM蛋白/Low calcium response locus protein Q蛋白/Recombinant Yersinia pseudotuberculosis serotype I Yop proteins translocation protein M (yscM)重组蛋白

    ￥69

    KTO: KIT A643 VALIDATION PROTOCOL (L)-Anatel A643a Accessories

    KTO: KIT A643 VALIDATION PROTOCOL (L)-Anatel A643a Accessories

    ￥50094

    PACS2抗体PACS2兔多抗抗体Phosphofurin acidic cluster sorting protein 2 antibody抗体PACS2 Antibody抗体

    PACS2抗体PACS2兔多抗抗体Phosphofurin acidic cluster sorting protein 2 antibody抗体PACS2 Antibody抗体

    ￥440

    相关问答

    问

    WB显出膜没有条带

    2924 围观
    2019-02-21

    问

    细胞增殖

    2521 围观
    2019-01-12

    问

    2小时能完成Western Blot实验，可能吗？

    2876 围观
    2019-01-07

    相关方法

    设 计 和 克 隆 一 个 sh R N A 到 慢 病 毒 沉 默 载 体 中 ：版 本 A

    设 计 和 克 隆 一 个 s h R N A 到 慢 病 毒 沉 默 载 体 中 ：版 本 B

    使 用 其 他 佐 剂 制 备 多 克 隆 抗 体 的 免 疫 接 种 法

    推荐阅读

    BACs, PACs, P1s and Plasmid Isolation Protocol (standard protocol)

    E.Z.N.A. Endo-Free BACs, PACs, P1s Protocol

    Determination of Plasmid Copy Number in Yeast

    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
    关注公众号
    反馈
    TOP
    打开小程序