PFGE n its mechanism as well application by shivendra kumar pg student at punjab agriculture university.

1. Pulse Field Gel Electrophoresis (PFGE)
(inventor-Schwartz and Cantor in 1984).
Name:-

2. What is pulse field gel
electrophoresis
• Pulsed-field gel electrophoresis (PFGE) is a
laboratory technique used by scientists to
produce a DNA fingerprint for a bacterial
isolate. A bacterial isolate is a group of the
same type of bacteria. PulseNet investigates
bacterial isolates from sick people,
contaminated food, and the places where
food is produced.

3. Instrumentation of PFGE

4. Why PFGE
• It is due to large size DNA fragments does
not penetrate the pores in agarose gel.
• Size of DNA fragments separated by :-

5. Procedure
Prepare bacterial isolates for pulsed-field gel electrophoresis
(PFGE) analysis by covering the bacteria in a substance called
agarose(molted)/plug mold,
Treated plugs are then loaded onto an agarose gel
Switch on the variable Electric field & DNA fragment get separated acc to
size.
Generate a DNA fingerprint and feed the data by software BioNumerics
Cell lysis to free the DNA in agarose plug followed by restriction
digestion.

6. How does PFGE work?

7. Protocol for PFGE
• Day 0 :- Incubate cultures at 37°C for 14-18 hours.
• Day 1 :- Making plugs :-.
• Turn on shaker water bath or incubator (54-55°C).
• Making the TE buffer (10 mM Tris:1 mM EDTA, pH
8.0):-1. 10 ml of 1 M Tris, pH 8.0
• 2. 2 ml of 0.5 M EDTA, pH 8.0
• 3. Dilute to 1lit with sterile Ultrapure Clinical
Laboratory Reagent Water (CLRW)

8. • (TE Buffer is used to make the plug agarose and
also to wash lysed PFGE plugs)
• 4. Prepare 1% agarose in TE Buffer (10 mM
Tris:1 mM EDTA, pH 8.0) for PFGE plugs as
follows:
• A . Weigh 0.50 g (or 0.25 g) agarose into 250 ml
screw-cap flask.
• B . Add 50.0 ml (or 25.0 ml) TE Buffer; swirl
gently to disperse agarose.
• C . Loosen or remove cap, cover loosely with
clear film, and microwave for 30 seconds;

9. • mix gently and repeat for 10 seconds intervals until
agarose is completely dissolved.
• D . Recap flask and return to 55-60°C/15min in water
bath and equilibrate the agarose or until ready to use.
• 4. Label small tubes (12mm x 75mm Falcon tubes or
equivalent) with culture numbers.
• 5. Prepare Cell Suspension Buffer (100 mM Tris:100
mM EDTA, pH 8.0) as follows:
• 5.1. 100 ml of 1 M Tris, pH 8.0
• 5.2. 200 ml of 0.5 M EDTA, pH 8.0
• 5.3. Dilute to 1000 ml with sterile Ultrapure water
(CLRW) .

10. • 6. Transfer 2 ml of Cell Suspension Buffer
(CSB) to small labeled tubes. Use a sterile
polyester-fiber or cotton swab that has been
moistened with sterile CSB to remove some of
the growth from agar plate; suspend cells in CSB
by spinning swab gently so cells will be evenly
dispersed and formation of aerosols is
minimized.
• 7. Adjust concentration of cell suspensions using
specrophotometer followed by adding additional
cells.

11. • Casting Plugs
• 1. Label wells of PFGE plug molds with culture number. When
reusable plug molds are used, put strip of tape on lower part of
reusable plug mold before labeling wells.
• 2. Transfer 400 µl adjusted cell suspensions to labeled 1.5-ml
microcentrifuge tubes.
• 3. Add 20 µl of Proteinase K (20 mg/ml stock) to each tube and
mix gently with pipet tip. (200 µl are needed for 10 cell
suspensions.)
• 4. Add 400 µl melted 1% agarose to 400 µl cell suspension; mix
by gently pipetting mixture up and down a few times.
• 5. Immediately, dispense part of mixture into appropriate well(s)
of reusable plug mold.
• . Allow plugs to solidify at room temperature for 10-15 minutes.
They can also be placed in the refrigerator (4°C) for 5 minutes

12. Lysis of Cells in Agarose Plugs
• using Cell Lysis Buffer (50 mM Tris:50 mM
EDTA, pH 8.0 + 1% Sarcosyl) and final vol
makeup 1 lit.
• Washing of Agarose Plugs After Cell Lysis.
• 1st
using lysis buffer.
• 2nd
using TE buffer.

13. Restriction Digestion of DNA in Agarose Plugs
• Using R.E
• For e.g :- for E.coli 0157 strain –
• primary enzyme- XbaI (50U/sample),
• secondary enzyme- BlnI/AvrII (30U/sample)
• tertiary enzyme- SpeI (30U/sample)
• Casting an Agarose Gel:
• Using electrophoretic buffer.TBE buffer of 0.5x
• 1% solidified agarose gel on to tank.

14. • Loading Restricted Plug Slices into the
Wells
• Staining and Documentation of an Agarose
Gel (Dilute 40 µl of ethidium bromide stock
solution (10 mg/ml) with 400 ml of
Ultrapure water ).
• Stain gel for 20-30 minutes in covered
container.
• Using destaining agent if too much
background is observed.
• Take the image……..

17. Electric field direction

19. PFGE
Advantage
• Band spreading is
minimized as variable
E.Field applied as well PH
gradient.
• High resolution achieved.
• Larger DNA fragments
could be identified.
• Protein that differs by as
little as 1/1000 PH that
separated.
Disadvantage
• As carrier ampholytes are in
high concentration results in
higher voltage
required(2000v).
• So for extra heat cooling is
necessary.

20. Application
• For separating proteins as well peptides
• In field of enzymology, cytology and in
taxonomy.
• For genome size estimation.
• For fingerprinting and physical mapping of
chromosome.
• In case of establishing degree of relatedness
among diff strain of same spp.

21. Practice
Restrict DNA in plugs
Slice 2mm piece of
plug
Load slices onto comb
Pour gel and remove comb

22. The Analysis Process: Normalization

23. E. coliE. coli DatabaseDatabase
• 308 isolates typed since 1999
• 170 pfge patterns
• 138 (44%) patterns observed only once
• Most common pattern (x.0002) seen 32
times = 10% database

24. Thank you