Deprecated: Required parameter $cat_id follows optional parameter $type in /www/wwwroot/ebimall.com/systems/hong.php on line 2088

Deprecated: Required parameter $where follows optional parameter $tree_id in /www/wwwroot/ebimall.com/systems/hlb.php on line 3505
PCR protocol188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
您好,欢迎您进入188进口试剂采购网网站! 服务热线:4000-520-616
蚂蚁淘商城 | 现货促销 | 科研狗 | 生物在线

PCR protocol

  • PCR reaction

    Protocol for 50µl reaction - adjust amounts if necessary, for a 20µl reaction use the same volumes of primer and dNTP-mix, but adjust the volume of water and 10x buffer, for a 100µl reaction use twice the volume of everything. This protocol is for with 10x buffer with added MgCl2, if this is not included add it to a concentration of 1.5 mM or other concentration found to be optimal.

  1. In a 200µl PCR tube take 5µl 10X PCR buffer (from enzyme kit).
  2. Add 1µl dNTP-mix.
  3. Add 1µl of 10 pmoles/µl solution of Forward primer.
  4. Add 1µl of 10 pmoles/µl solution of Reverse primer.
  5. Add 1µl template DNA, or a tiny bit of colony on agar-plate picked with a tooth-pick.
  6. Add 40.5µl (or 41.5µl if using a colony pick) dH2O.
  7. Add 0.5µl Taq DNA polymerase (5U/µl).
  8. Mix gently by pipetting.
  9. Place in PCR machine, and close hot-lid (if the machine does not have a hot-lid add a couple of drops of mineral oil on top of reaction to prevent evaporation.
  10. Perform PCR with a suitable program for the template, primers, and thermal cycler:
    • 5 minutes at 95°C (especially important if using colony pick or unopened cells af any form)
    • 30 times:
    • 30 seconds at 95°C (depending on PCR machine)
    • 30 seconds at 50°C (adjust according to annealing temperatures of primers)
    • 30 seconds at 72°C (long PCR products (>1kb) require longer)
    • 6 minutes at 72°C

    Agarose gel for analysis of PCR products

    Protocol for 60 ml gel - adjust amounts if necessary.

  11. In a 250ml conical bottle take 0.72g of agarose.
  12. Add 60 ml of water.
  13. Heat to boiling in microvawe owen.
  14. Cool down to 60�C.
  15. Add 2µl 10 mg/ml ethidium bromide.
  16. Pour gel in tray with tape at ends and 8 tooth comb inset.
  17. Allow to set.
  18. Take 10µl of the PCR reaction and place on a piece of Saran wrap.
  19. Add 2µl 6X loading buffer.
  20. Load on gel along with molecular weight marker.
  21. Run at 100V until dye front has reached approximately 3/4 down the gel.
  22. Inspect under UV.
dNTP-mix for PCR
10µl 100mM dATP, 10µl 100mM dCTP, 10µl 100mM dGTP, 10µl 100mM dTTP, 60µl DEPC-treated dH2O
6X loading buffer for agarose gel
25µl 1% (w/v) bromo-phenol blue, 25µl 1% (w/v) xylene-cyanol FF, 30µl 100% glycerol, 20µl dH2O


新闻动态
行业前沿
技术文章
最新产品

188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap