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

    Mag-Bind Blood RNA Isolation Protocol (for 50ul Blood)

    互联网

    1087

    实验原理

    If using the Mag-Bind™ Blood RNA Kit for the first time, please read this booklet to become familiar with the procedure and its various modifications. Samples are lysed in a specially formulated buffer containing detergent and chaotropic salt. After adjust the buffer condition, nucleic acids (DNA/RNA) will form a complex with magnetic beads. The beads/nucleic acids complex is separated from lysates using a magnet. Proteins and cellular debris are efficiently washed away by a washing step. Next, DNA is removed by DNase I treatment. RNA is re-bound and cleaned from the DNase I reaction mixture using a second magnetic beads binding and washing procedure. Finally, pure RNA is then eluted in nuclease-free water or low ionic strength buffer. Purified RNA can be directly used in downstream applications without the need for further purification.

    实验试剂

    Regents supplied by User

    1. Absolute ethanol (96-100%)

    2. Isopropanol

    实验设备

    Equipments supplied by User

    1. Magnetic separation device for 96 well microplate (MSD-01)

    2. Nuclease-free 96-well

    3. Multichannel pipette

    4. Nuclease-Free pipetting tips

    实验步骤

    The following protocol is designed for isolating total RNA from Fresh Whole Blood. For best RNA quality, always use blood that has not been frozen. Frozen blood can be used with this protocol, however, RNA quality could be compromised as the results of freezethawing process.

    1. Prepare a Lysis Mixture by mixing one volume of Buffer MRB and one volume of isopropanol. For 96 samples, combine15 ml Buffer MRB with 15 ml isopropanol and mix throughly by vortexing. Add 300 ul Lysis Mixture for each sample at step 3.

    2. Add 50 ul blood sample into Processing Plate (supplied) followed by adding 10 ul Proteinase K solution and 10 ul Mag-Bind™ Particle R.

    Note: Proteinase K and Mag-Bind™ Particles R can be prepared as master mix and add to each at one time. For 96 samples, mix 1 vial Mag-Bind™ Particle R (1 ml) with 1 ml Proteinase K solution. Add 20 ul beads/proteinase K mix for each blood sample.

    3. Add 300ul Lysis Mixture, shaking for 10 minutes at room temperature.

    4. Place the Processing Plate on a magnetic separation device to magnetize the magnetic particles. Leave the plate on the magnet until all the magnetic particles are pelleted. The capture time is depend on the sample type and magnetic stand used. If Omega Bio-tek MSD-01 is used, it normally take 7-15 minutes.

    5. Carefully aspirate and discard the supernatant without disturbing the magnetic particles.

    6. Remove the Processing Plate containing the magnetic particles from the magnetic separation device. Add 300 ul of Buffer MBW and resuspend magnetic particles pellet by pipetting up and down or vortex the plate at maximum speed for 1 minute.

    7. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate and disacrd the cleared supernatant after the lysate is cleared.

    8. Remove the plate containing the magnetic particles from the magnetic separation device. Add 400 ul of SPR Wash Buffer to each well and resuspend magnetic particles pellet by pipetting up and down or vortexing for 30 seconds.

    9. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate an discard the cleared supernatant after the lysate is cleared.

    10. Leave the tube on the magnetic separation device. Prepare the DNase I digestion mix as following table.

    Note: If total nucleic acid (DNA and RNA) is desired, skip this DNase I digestion steps (step 14-21) and proceed step 17 for isolating both DNA and RNA.

    Note: If total nucleic acid (DNA and RNA) is desired, skip this DNase I digestion steps (step 14-21) and proceed step 17 for isolating both DNA and RNA.

    11. Add 50 μl of DNase I digestion mix and resuspend the magnetic particles by pipetting up and down for 20 times or vortexing. Incubate at room temperature for 10-15 minutes.

    Note: It is very important to remove any liquid drop from the wells of the process plate before adding the DNase I digestion mix. DNase I digestion Mix must be used immediately once it is prepared.

    12. Add 300 μl Buffer MBW and mix throughly by vortexing for 3 minutes. Incubate at room temperature for 5-10 minutes.

    13. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate the cleared supernatant after the lysate is cleared.

    14. Add 400 ul of SPR Wash Buffer and resuspend magnetic particles pellet by pipetting up and down for 20 times or vortexing at maximum speed for 30 seconds.

    15. Place the plate onto a magnetic separation device to magnetize the Mag-Bind™ particles.

    16. Aspirate and discard the cleared supernatant. Carefully remove any liquid drop from tube. Leave the tube on the magnet stand. Air dry the magnetic particles at room temperature for 7-10 minutes.

    17. Add 20-50 μl DEPC Treated water and resuspend magnetic particles pellet by vortexing or pipetting up and down for 20 times. Incubate at room temperature for 3 minutes.

    18. Place the tube onto a magnetic separation device to magnetize the Mag-Bind. particles.

    19. Transfer the cleared supernatant contains purified RNA into a new RNase-free microplate .

    相关产品推荐

    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

    Bind-Silane 亲和硅烷

    Bind-Silane 亲和硅烷

    ￥188

    ///蛋白Recombinant Dermatophagoides farinae Tropomyosin重组蛋白Mag44 Allergen: Der f 10蛋白

    ///蛋白Recombinant Dermatophagoides farinae Tropomyosin重组蛋白Mag44 Allergen: Der f 10蛋白

    ￥2328

    NEST 50uL导电吸头，盒装

    NEST 50uL导电吸头，盒装

    ￥4800

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

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

    ￥50094

    相关问答

    问

    WB显出膜没有条带

    3011 围观
    2019-02-21

    问

    细胞增殖

    2568 围观
    2019-01-12

    问

    2小时能完成Western Blot实验，可能吗？

    2986 围观
    2019-01-07

    相关方法

    使用 Trizol 纯化 RNA 实验

    利用 Marligen 总 RNA 快速纯化系统纯化总 RNA 实验

    用T4 RNA 连接酶连接 RNA 分子

    推荐阅读

    E.Z.N.A. Mag-Bind Blood DNA Protocol (1-100 ul Blood)

    Mag-Bind Blood DNA Maximum Yield Protocol (2ml-6ml)

    Mag-Bind Blood DNA Maximum Yield Protocol (7ml-10ml)

    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