酵母菌落PCR

   简介:前段时间本人做了酵母PCR实验,几经周折,终于做出来了,先将本人收集的几种酵母PCR方法介绍如下.

1. 冷泉港实验室提供的经典方法,摘自<酵母遗传学方法实验指南>(Methods in Yeast Genetics: A Cold Spring Harbor
   Laboratory Course Manual)(致谢华中科大杨洋老师)
  1)在冰上配制反应混合液：
        2μl         10×菌落PCR缓冲液
        1.2μl      25mmol/L MgCl2
      0.4μl      10mmol/L dNTPs
      10pmol      引物（每一种引物）
        0.2μl      Taq DNA聚合酶（5U）
        加水到总体积为20μl
  2)用移液器加微量细胞（约0.25μl）到PCR反应混合液中（20μl反应体积）
  3)PCR循环参数：
           94℃ 4分钟
           94℃ 1分钟
           55℃ 1分钟
           72℃ 2分钟
           35个循环    ;
           72℃ 10分钟
  4)上全部样品进行琼脂糖电泳。

2. 网络上提供的一个改进版本.由于酵母细胞壁教厚,上述经典方法效果欠佳,这种方法利用SDS帮助破壁利于基因组DNA的释放

        This method is more reliable than the old method of adding yeast directly to PCR tubes. No more than 1 microliter of the crude DNA should be added to the 50 microliter PCR reaction because the SDS in the DNA prep can inhibit the PCR reaction.
      Note: These elongation times and annealing temperatures work well for most applications where
the product is less than 1.0 kb. These parameters may have to be adjusted for your specific application. Platinum Taq (or other hot start Taq) gives more reliable and consistent results than regular Taq for this assay.
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1) Prepare Yeast DNA (this works best with fresh yeast plates)
   Use a pipetman tip to transfer the equivalent of a medium size yeast colony to 30 microliters of 0.2% SDSVortex ~15 seconds .Heat in hot block for 4 min at 90 deg. (important for consistent DNA extraction and PCR results)Spin in microfuge 1 min. Remove supernatant to a new tube. The crude DNA can be stored at -20 degrees.
2) PCR Reaction
  Combine the following components on ice:
  5 microliters 10x colony PCR buffer
  1.5 microliters 50 mM MgCl2
  1 microliter 10 mM mix of dNTPs
  10 pmoles of each primer (~100 ng of a 25 mer oligo)
  2 microliters 25% Triton X-100
  0.3 microliter Platinum Taq Polymerase [Invitrogen] (5 u/microliter)
  H2O to a final volume of 49 microliters
Add mix to PCR tubes on ice containing 1 microliter of the crude DNA prep from above
3) PCR cycle profile:
   95 deg 1 min
   95 deg 30 sec
   54 deg 1 min (optimum annealing temp varies according to primiers)
   72 deg 1 min
   repeat steps 2 through 4 for a total of 35 cycles
   72 deg 6 min
   hold at 4 deg
     Load 5-10 microliters to Agarose gel for assay.

  Warning:For DNA sequencing analysis of product, purify using QIAquick PCR purification kit
   (Qiagen), eluting the product in 30 microliters. Use ~6 microliters for DNA sequencing analysis.