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[Bio101] Pulsed-field Gel Electrophoresis Typing of Gram-negative Bacteria (E.coli)
[Bio101] 革兰氏阴性菌的脉冲场凝胶电泳分型(大肠杆菌)   

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Abstract

Keywords: Pulsed field gel electropheresis(脉冲场凝胶电泳), Gene typing(基因分型), E.coli(大肠杆菌), Gram negative bacteria(革兰氏阴性菌)

Materials and Reagents

  1. Chromosomal low melting agarose (Lonza InCert Agarose or Bio-Rad Laboratories)
  2. Restriction enzymes (New England Biolabs)
  3. Proteinase K (Promega Corporation) 
  4. 10x Tris-borate EDTA (TBE) buffer (Promega Corporation)
  5. Agarose for pulsed field gel (Bio-Rad Laboratories) 
  6. Lamda ladder for pulsed field gel (Bio-Rad Laboratories)
  7. Ethidium bromide (10 mg/ml) (Life Technologies, Invitrogen™)
  8. Agar media
  9. TE
  10. NaCl
  11. EDTA
  12. Sarkosy l
  13. Sma I
  14. Xba I
  15. Restriction enzyme buffer
  16. Running buffer (0.5x TBE)
  17. Suspension buffer (see Recipes)
  18. Lysis buffer (see Recipes)

Equipment

  1. CHEF-MapperTM apparatus (Bio-Rad Laboratories)
  2. Spectrophotometer (Bio-Rad Laboratories) 
  3. UV light and photograph
  4. Water bath
  5. Glass rod
  6. 1.5 ml Eppendorf tube

Procedure

  1. Chromosomal DNA preparation:
    1. Subculture strains onto agar media. Grow under appropriate conditions for 1-3 days.
    2. Harvest cells and suspend into 2 ml of suspension buffer. Adjust cell density to OD600: 1.5 to 2.0. Standard cell OD ensures that each sample contains approximately the same amount of DNA.
    3. Prepare 1.2% chromosomal grade low melting agarose for making bacterial DNA plugs and cool it to 56 °C in water bath. 
    4. Add 200 μl molten chromosomal grade agarose suspensions to 200 μl of bacterial suspension. Mix and dispense into 3 chambers of the mould. Then place the plug in the fridge (4 °C) for about 10 min until the gel is solidified.
    5. Remove agar blocks by inserting a blunt-tipped glass pipette and place each set of 3 plugs into a 10 ml plastic tube containing 2 ml lysis buffer. Add 1 mg/ml Proteinase K and incubate at 55 °C overnight. This leaves only DNA embedded in the solid agar matrix. The DNA will not shear when handled further and remains stable for analysis by electrophoresis.
    6. Discard lysis buffer from samples and wash four times, each for at least 20 min periods, in 2 ml of 10:1 TE (10 mM Tris-1 mM EDTA) buffer at 55 °C. Store plugs at 4 °C.

  2. DNA digestion:
    1. Remove an agar/DNA plug using a glass rod and place it into a 1.5 ml Eppendorf tube. Add 300 μl restriction enzyme buffer and incubate for 30 min at room temp. Incubation in digestion buffer to makes DNA more amenable to digestion.
    2. Remove buffer and add 250 μl of restriction enzyme buffer which contains 20 units of enzyme (Sma I or Xba I) for 5 h at 37 °C.

  3. DNA electrophoresis:
    1. Boil 100 ml 1.0% pulsed gel agarose in microwave for separation of DNA fragments. Cool in water bath to 50-56 °C.
    2. Assemble gel mould and pour gel into mould. 15 or 30 (Bio-Rad equipment) well-forming comb must be inserted into the assembly before the agarose is poured in.
    3. Remove gel well-forming comb and slide DNA plugs into wells using a clean plastic scalpel (be sure that each slice is straight and adheres onto the front side of the well). Also add lambda ladder into the first, last and middle wells fragments.
    4. Add 2 L running buffer (0.5x TBE) to electrophoresis chamber and run through pump and cooling unit (14 °C) about 30 min before use. Place gel into electrophoresis tank and close cover.
    5. Set electrophoresis conditions on power pack and computer. Program run according to the instructions under two-state mode, and start run.
      Block 1: angle: 60+, 60-; pulse time: 1 sec to 30 sec; ramping factor: 0(linear); run time: 17 h; voltage gradient: 6.0 V/cm.
      Block 2: 5 sec to 9 sec for 6 h, the rest is the same as the block 1.

  4. Staining and visualizing the gel:
    1. Switch off electrophoresis unit. Remove gel from base and gently place into 400 ml, 0.5x TBE buffer with 40 µl ethidium bromide. Stain 30 min, distain with water for 5 min.
    2. View under UV light & photograph. Make sure the top of the MW marker should always be visible and outer edges of the first and last samples are in the printed image.

Recipes

  1. Suspension buffer
    1 M NaCl
    10 mM Tris (pH 8)
    10 mM EDTA
  2. Lysis buffer
    0.5 M EDTA
    1% Sarkosy l

简介

关键字:脉冲场凝胶电泳, 基因分型, 大肠杆菌, 革兰氏阴性菌

材料和试剂

  1. 染色体低熔点琼脂糖(Lonza InCert Agarose或Bio-Rad Laboratories)
  2. 限制酶(New England Biolabs)
  3. 蛋白酶K(Promega Corporation)
  4. 10x Tris-硼酸盐EDTA(TBE)缓冲液(Promega Corporation)
  5. 用于脉冲场凝胶的琼脂糖(Bio-Rad Laboratories)
  6. 脉冲场凝胶(Bio-Rad Laboratories)
  7. 溴化乙锭(10mg/ml)(Life Technologies,Invitrogen TM)
  8. 琼脂培养基
  9. TE
  10. NaCl
  11. EDTA
  12. Sarkosy l
  13. I
  14. Xba I
  15. 限制性酶缓冲液
  16. 运行缓冲液(0.5x TBE)
  17. 悬浮缓冲液(见配方)
  18. 裂解缓冲液(见配方)

设备

  1. CHEF-Mapper TM装置(Bio-Rad Laboratories)
  2. 分光光度计(Bio-Rad Laboratories)
  3. 紫外光和照片
  4. 水浴
  5. 玻璃棒
  6. 1.5 ml Eppendorf管

程序

  1. 染色体DNA制备:
    1. 亚克隆培养物在琼脂培养基上。 在适当的条件下生长1-3天。
    2. 收获细胞并悬浮于2ml悬浮液缓冲液。将细胞密度调节至OD 600:1.5至2.0。标准细胞OD确保每个样品含有大约相同量的DNA。
    3. 准备1.2%染色体级低熔点琼脂糖用于制作细菌DNA插头,并在水浴中冷却至56°C。
    4. 添加200微升熔融染色体级琼脂糖悬浮液200微升细菌悬浮液。混合并分配到模具的3个室中。然后将塞子放在冰箱(4℃)中约10分钟,直到凝胶凝固。
    5. 通过插入钝头玻璃吸管除去琼脂块,并将每组3个插头放入10毫升塑料管,含有2毫升裂解缓冲液。加入1mg/ml蛋白酶K,并在55℃下孵育过夜。这仅留下嵌入固体琼脂基质中的DNA。当进一步处理时,DNA不会剪切,并且对于通过电泳分析保持稳定。
    6. 从样品中弃去裂解缓冲液,并在55℃下在2ml 10:1 TE(10mM Tris-1mM EDTA)缓冲液中洗涤四次,每次至少20分钟。将插头存放在4°C。

  2. DNA消化:
    1. 使用玻璃棒移除琼脂/DNA插塞,并将其放入1.5毫升的Eppendorf管。 加入300μl限制酶缓冲液,并在室温下孵育30分钟。 在消化缓冲液中孵育以使DNA更易于消化。
    2. 取出缓冲液,并在37℃下加入250μl含有20单位酶(Sma I或Xba I)的限制酶缓冲液5小时。

  3. DNA电泳:
    1. 沸腾100毫升1.0%脉冲凝胶琼脂糖在微波中分离DNA片段。 在水浴中冷却至50-56℃。
    2. 组装凝胶模具并将凝胶倒入模具中。 15或30(Bio-Rad设备)孔形成梳子必须在倒入琼脂糖之前插入组件中。
    3. 取出凝胶形成梳子,并使用干净的塑料手术刀(确保每个切片是直的,并粘附到井的前侧)的DNA插入井。还将λ梯子添加到第一,最后和中间孔片段中。
    4. 加入2 L运行缓冲液(0.5×TBE)到电泳室,运行通过泵和冷却单元(14°C)约30分钟后使用。将凝胶放入电泳槽并盖上盖子。
    5. 在电源包和计算机上设置电泳条件。程序根据双态模式下的指令运行,并开始运行。
      块1:角度:60+,60-;脉冲时间:1秒至30秒;斜率因子:0(线性);运行时间:17小时;电压梯度:6.0V/cm 块2:5秒至9秒持续6小时,其余与块1相同。

  4. 染色和可视化凝胶:
    1. 关闭电泳装置。 从碱中除去凝胶,轻轻放入400 ml,0.5x TBE缓冲液中,加入40μl溴化乙锭。 染色30分钟,用水蒸馏5分钟。
    2. 在UV光& 照片。 确保MW标记的顶部始终可见,并且第一个和最后一个样品的外边缘在打印的图像中。

食谱

  1. 暂停缓冲区
    1 M NaCl
    10mM Tris(pH8)
    10 mM EDTA
  2. 裂解缓冲液
    0.5 M EDTA
    1%Sarkosy l
  • English
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Z. (2011). Pulsed-field Gel Electrophoresis Typing of Gram-negative Bacteria (E.coli). Bio-protocol Bio101: e103. DOI: 10.21769/BioProtoc.103;
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upasana pathak
hnmrs
Hello,
can you please let me know the substitute I can use instead of 1% Sarkosy l in lysis buffer.
also as I am a beginner , it will kind of you if you can suggest modifications to the protocol when genotyping P.aeruginosa and Acinetobacter baumanii.

Thanks
10/12/2016 12:36:31 AM Reply
姜 云栋
中国农业大学
good
3/21/2012 11:00:21 AM Reply