脉冲场凝胶电泳

PFEG（Plused-Field Gel Electrophoresis）技术是将细菌完全包裹在一种特殊的琼脂中，然后加入一些化学试剂，比如十二烷基肌氨酸钠，蛋白酶K等，将细菌中的蛋白成分全部溶化，消化，只剩下整个序列的DNA。经稀有限制性内切酶消化后，切割成大小不等的片断，然后将其置于脉冲场电泳槽中电泳，完成片段分离的过程。

经染色脱色后，在读胶仪上显现酶切后的电泳图谱，并经统计软件的分析，即可判断出条带的不同，同时做出细菌的同源性分析，从而达到分型的目的。其中能影响电泳图谱的参数很多，如电泳的转换时间，电泳的总时间，电泳的角度，电压，电泳液的温度等等，且不同的参数会得到不同的电泳图谱。

Pulsed Field Gel Electrophoresis Short Protocol

1. Reagents

1.1 Tris-EDTA Buffer （10mM Tris： 1mM EDTA， pH 8.0）.

- 10ml of 1M Tris， pH 8.0

- 2ml of 0.5M EDTA， pH 8.0

- Dilute to 1000ml with molecular grade water.

1.2 Cell Suspension Buffer （CSB）. （100mM Tris：100mM EDTA， pH 8.0）.

- 10ml of 1M Tris， pH 8.0

- 20ml of 0.5M EDTA， pH 8.0

- Dilute to 100 ml with molecular grade water.

1.3 InCert agarose for plugs （1.6% InCert： 1% SDS agarose in TE）.

- Weigh 0.8g InCert agarose into a 250 ml screw-cap flask

- Add 46.7 ml TE Buffer； swirl gently to disperse agarose.

- Microwave for 30 sec.， mix gently and repeat for 10 sec.

- Place flask in 55-60℃ water bath for 5 min before adding SDS.

- Add 2.5 ml of 20% SDS and mix well.

- Keep at 55-60oC water bath until ready to use.

1.4 Cell Lysis Buffer （50mM Tris：50mM EDTA， pH， 8.0 + 1% Sarcosine）.

- 25 ml of 1M Tris， pH 8.0

- 50ml of 0.5M EDTA， pH 8.0

- 50ml of 10% N-Lauryl Sarcosine.

- Dilute to 500 ml with sterile type 1 water.

1.5 Seakem Gold agarose， SKG （ in 0.5X TBE）.

- Weigh 2g SKG in to 500 ml screw-cap flask containing 200 ml 0.5XTBE.

- Swirl gently to disperse agarose.

- Remove cap， cover loosely with clear film， and microwave for 60-sec.

- Mix gently and repeat for 15 more seconds till agarose dissolves.

- Recap flask and place in 50-60℃ water bath.

2. Isolation and embed allowfullscreen='true'ding of cells in agarose

2.1 Streak isolated colony to agar plates for a confluent growth. Incubate cultures at 37℃ for 14-18 hrs.

2.2 Transfer 2 ml of CSB to small labeled tubes.

2.3 Remove some of the growth from the agar plate using sterile cotton swab and suspend cells gently in CSB so that cells will be evenly dispersed and formation of aerosols minimized.

2.4 Adjust cell suspension to 20% transmittance using Biomerieux （Vitek）colorimeter.

2.5 Transfer 200 μl suspension to labeled 1.5 ml sterile microfuge tubes using p1000 pipette to reduce DNA shearing.

2.6 Add 10μl of Proteinase K （20mg/ml stock） to each tube and mix gently by inverting tubes 5 to 6 times.

2.7 Add 200 μl melted 1.6% InCert to the 200 ul of cell suspension； mix gently by pipetting up and down gently few times. Avoid creating air bubbles.

2.8 Immediately dispense part of mixture into disposable plug mold. Do not allow air bubbles to form.

2.9 Allow plugs to solidify at room temperature （10-15 min.） or 5 min in 4℃.

3. Lysis of cells in agarose plugs.

3.1 Transfer plugs to appropriately labeled 2ml round bottom tubes.

3.2 Measure 1.5 ml cell lysis buffer X the number of tubes.

3.3 Add 40 ul of Proteinase K solution （20mg/ml） X the number of tubes to the cell lysis buffer.

3.4 Add 1.5 ml of Proteinase K/Cell lysis buffer to each 2 ml round bottom tube.

3.5 Place tubes in a rack and incubate in a 54C shaker water bath for 2 hours with constant and vigorous agitation. Be sure water level is above the lysis buffer level.

3.6 Preheat enough sterile water and TE to 50℃ for the next step.

4. Washing of agarose plugs after cell lysis

4.1 After lysis， remove tubes from water bath and transfer plugs to 50 ml tubes.

4.2 Add at least 10 ml of sterile water preheated to 50℃ to each tube and shake the tubes vigorously in water bath for 15 minutes.

4.3 Pour off water and repeat wash one more time.

4.4 Pour off water and add at least 10 ml preheated TE. Shake the tubes vigorously in a 50℃ water bath for 15 minutes.

4.5 Pour off TE and repeat wash step with pre-heated TE 3 more times.

4.6 Decant last wash and add 5 ml sterile TE. Plugs can be stored at 4℃ in TE until restriction digestion is done.

5. Restriction digestion of DNA in agarose plugs

5.1 Label 1.5 ml microfuge tubes with culture numbers.

5.2 Prerestriction incubation

- Dilute 10X H buffer （Roche） 1：10 with molecular grade water.

- Add 150ul 10X Hbuffer to 1350 ul water for 15 plug slices.

5.3 Carefully remove plug from TE with spatula and place it on petriplate

5.4 Cut two 1mm slices with sterile scalpel and transfer to tube containing diluted H-buffer.

5.5 Incubate sample plug slices in 37℃ water bath for 5-10 min or at room temperature for 15 min.

5.6 Remove buffer from plug slices using pipette tip all the way to the bottom.

Be careful not to cut plugs with the tip of pipette.

5.7 Dilute 10X H buffer 1：10 with molecular grade water and add Xba1 resteriction enzyme.

- For 15 plug slices， add water 1305 ul， 10X H buffer， 150 ul and enzyme 45 ul （of 10U/ul）.

5.8 Add 100 ul of restriction mixture to each tube. Close tube and mix by tapping gently.

5.9 Incubate sample and control plug slices in 37℃ water bath for 2 hours.

6. Casting agarose gel

6.1 Equilibrate water bath to 50-60℃.

6.2 Make 2500 ml of 0.5X TBE （by mixing 250 ml of 10X TBE with 2250 ml of water）.

6.3 Cool melted SKG agarose in 50-60C water bath for 5-6 min.

6.4 Carefully， pour agarose into gel form fitted with comb. Be sure， there are no bubbles.

6.5 Put black gel frame into CHEF DR chamber and add 2.0-2.2 l fresh 0.5X TBE.

6.6 Turn on cooling module （14℃）， power supply， and pump setting at 70 for a flow of 1 l /min. 30min. before gel is run.

6.7 Remove comb from gel after it solidifies.

6.8 Remove restricted plug slices from tubes with spatula and load into appropriate wells. Manipulate position with spatula to the bottom and front of wells.

Be sure there are no bubbles.

6.9 Fill in wells of with melted SKG agarose.

6.10 Carefully place gel inside black gel frame in electrophoresis chamber and close cover of chamber.

7. Electrophoresis conditions

For CHEF DR II or III

- Initial A time： 2.2 sec - Voltage： 6 V/cm

- Final A time： 63.8 sec. - Run time： 18 hours.

8. Staining and documentation of PFGE agarose gel

8.1 Turn of equipment， remove and stain gel with ethidium bromide by diluting 40 ul of EtBr stock solution （10mg/ml） with 400 ml of reagent grade water.

8.2 Stain gel for 20-30 min. in covered container.

8.3 Destain gel in approximately 500 ml reagent grade water for 60-90 min；change water every 20 min.

8.4 Capture image on Gel Doc 2000， or equivalent documentation system.

8.5 Save image as .tif file and analyze data using using Quantity one orequivalent software.