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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)

微生物学 > 微生物遗传学 > DNA > 染色体
作者: Zhenying Liu
8/5/2011, 5968 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.103

[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)

[Abstract]

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

材料和试剂

 

1.       染色体低熔点琼脂糖(Lonza InCert Agarose or Bio-Rad

2.       限制性内切酶(New England Biolab

3.       蛋白酶K(Promega)

4.       10倍的Tris - EDTA的硼酸盐(简称TBE)缓冲液(Promega公司)

5.       脉冲场琼脂糖凝胶(Bio-Rad

6.       脉冲电泳分子标记(Bio-Rad

7.       溴化乙锭(10 mg/ml)(Invitrogen

 

实验设备

 

1.       脉冲场凝胶电泳仪

2.       分光光度计

 

步骤

 

1.      染色体DNA样品的制备

1)      在培养基上继代培养的菌株,在适宜的条件下培养1-3 d

2)      收集菌体悬浮于2 ml的缓冲液中,调整细胞密度为OD6001.5-2.0。标准的OD值应确保每个样本中包含大致相同数量的DNA

3)      制备1.2%的低熔点琼脂糖凝胶,水浴冷却至56°C,用于制胶;

4)      将等体积(各200μl)菌体悬液与琼脂糖溶液混匀,立即等分流至模具的3个模块中。于4°C冰箱中冷却10 min直至胶块凝固;

5)      插入一钝头的玻璃吸管将凝胶块取出放入含有2 ml裂解液的10 ml塑料管中。加入1 mg/ml蛋白酶K55°C培养过夜。仅DNA嵌入到固体琼脂块中,在进一步处理时不会断裂,电泳分析时保持稳定;

6)      弃掉裂解液。在55°C条件下,用2 ml TE缓冲液(10mM Tris-1mM EDTA)清洗4次,每次20 min。置于4°C保存。

2.      DNA消化

1)      用玻璃棒将DNA/琼脂糖凝胶块移出,置于1.5 ml离心管中。加入300 μl限制性内切酶缓冲液,室温放置30 min。置于消化缓冲液中DNA更易消化。

2)      弃去缓冲液,加入250 μl限制性内切酶缓冲液,其中含有20 U的酶(SmaIXbaI),37°C温育5 h

3.      DNA电泳

1)          制备100 ml 1.0%的琼脂糖凝胶用于分离DNA片段,水浴冷却至50-56°C

2)          组装胶模并制胶。插入1530孔齿梳(Bio-Rad设备),制胶。

3)          小心拔出齿梳,用干净的塑料手术刀修整DNA凝胶块(确保每个削面笔直并保持正面向上),在样品片段第一、最后或中间添加DNA分子量标记;

4)          电泳槽中加入2 L的电泳液,缓冲液电泳前14冷却30 min。把胶放入电泳槽内,盖上盖子;

5)          通过电源和电脑设置电泳条件。程序根据一个二态模型下的指令运行。

6)          程序:电泳角度:60+60-;脉冲时间:1 S30 S;缓冲因子:0(线性);运行时间:17 h;电压梯度:6.0 V/cm

程序二:脉冲时间5S9S运行6 h,其余同一。

4.      凝胶染色和观察分析

1)          关闭电泳。轻轻的将胶块移到含有40 μl 溴化乙锭的400 ml 0.5×TBE缓冲液中。染色30 min,水中显色5 min

2)          凝胶成像。确保MW标记的最上面清晰可见,第一个和最后一个样品的边缘处于照片中。

 

配方

 

1.       悬浮缓冲液:1 M NaCl10 mM Tris pH 810 mM EDTA

2.       裂解缓冲液:0.5 M EDTA+1%肌氨酰 Sarkosyl

 

 

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How to cite this protocol: Liu, Z. (2011). Pulsed-field Gel Electrophoresis Typing of Gram-negative Bacteria (E.coli). Bio-protocol Bio101: e103. DOI: 10.21769/BioProtoc.103; Full Text



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10/12/2016 12:36:31 AM  

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

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3/21/2012 11:00:21 AM  

姜 云栋
中国农业大学

good

Reply

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