Blackburn:Yeast Colony PCR v2.0 protocol - Op...

来源：百度文库 编辑：神马文学网 时间：2024/04/20 07:36:01

Blackburn:Yeast Colony PCR v2.0 protocol

From OpenWetWare

Jump to: navigation, search

Solutions/reagents:

  • 0.02M NaOH
  • Q-solution
    (5X)
  • PCR buffer
    (10X)
  • dNTPs
    (10mM each)
  • Forward primer
    (100µM)
  • Reverse primer
  • Taq
  • ddH2O
  • a small colony

Equipment:

  • Thermocycler
  • Sterile 0.6-ml tubes

Steps:

  1. Yeast Cell Lysis

    1. Measure out 10 µl of 0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1).

      2.  

    2. Add a small colony.
      Resuspend pellet by vortexing/by shaking vigorously.
      If the solution is cloudy, you've added enough cells.
      I have been told adding too much yeast can inhibit the reaction.

      3.  

    3. Set the thermocycler to run the following program:
      • 99°C, 10 mins
      In the mean time, prepare the master mix for the PCR reaction.
      The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.

      4.  

    2.  

  2. PCR

    1. Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):

        Q-solution PCR buffer dNTPs Forward primer Reverse primer Taq ddH2O Colony PCR 2 µl 1 µl 0.2 µl 0.2 µl 0.2 µl 0.1 µl 5.3 µl

      2.  

    2. Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot.
      Measure out 9 µl of master mix solution into Master Mix aliquot.

      3.  

    3. Add 1 µl of boiled sample.
      A multichannel pipette is helpful here.

      4.  

    4. Program a standard thermocycler to run the reaction using the following parameters:
      Initial denaturation
      • Denature: 95°C, 5 mins
      Thermocycling
      • No. of cycles: 30
      • Denature: 95°C, 10 secs
      • Anneal: 50°C, 10 secs
      • Elongate: 72°C, 60 secs
      Elongation time : 1 min/kbp.I generally do 30 sec elongation.
      Termination
      • Elongate: 72°C, 10 mins
      • Hold: 4°C, until removed from machine
      I don't think this step is critical.

      5.  

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 6 mins

Blackburn:Yeast Colony PCR v2.0 protocol - Op... 古玉雕欣赏0op 数据库设计规范V2.0 Lightbox JS v2.0 [1] Lightbox JS v2.0 [2] MIDP v2.0规范简介 MIDP v2.0规范简介 TS PCR PCR注意事项 Protocol Overhead memcached protocol Top 100 web2.0网站V2.0 Top 100 web2.0网站V2.0 卡巴KEY挖掘机 V2.0 中文绿色版 U盘巡警 V2.0 build 0330 迅雷VIP帐号获取器 V2.0 TAIL-PCR(Termal Asymmetric Interlaced PCR) Dutch colony islands get new status PCR的物理意义 PCR的物理意义 关于Touchdown PCR SiteFinding-PCR啊 xmlBlaster - Requirement protocol.email Network Time Protocol