PFGE:General Overview and Troubleshooting Tips

Kara Cooper, Ph.D.PulseNet Methods Development and Reference Laboratory

Enterics Disease Laboratory BranchCenters for Disease Control and Prevention

PFGE Process

SpecimenPatient Specimen

Collection

+

CellSuspension

Agarose

=

Cells Trappedin Plug

Cell Lysis and Plug Washing

Restriction Electrophoresis Imaging

Grow isolatedcolony

US Dime coin

Components of Agarose Plugs

Overnight bacterial growthAdjust cell concentration in Cell Suspension Buffer

(100 mM Tris, 100 mM EDTA, pH 8.0, 1% Sarcosyl)

Proteinase K (0.5 mg/ml) treatment of cell suspension inactivates endogenous nucleases such as RNases and DNases

Casting plugs to ensure the isolation of intact DNA molecules

• Reusable or disposable plug molds• 1% SeaKem Gold • SDS is not necessary for most organisms

• Undue stress to bacterial cells prior to casting the plug can cause premature cell lysis resulting in DNA degradation (smearing)

• Important to:– Grow cultures on non-selective media– Plugs should be made from fresh cultures

(14-18 hours old) – DO NOT centrifuge or vortex cell

suspensions– DO NOT leave bacteria in cell suspension

mixture for extended periods of time prior to casting plugs

48 hrs18 hrs

Plug Preparation

Plug Preparation

• Cell suspension concentration– Band intensity is relatively similar

from cell suspensions of 0.3 to 0.5 (as measured with Dade Microscan turbidity meter)

– Fewer cells = More efficient lysis = Similar band intensity

– Benefits of lower cell suspension:• Sharper bands• Increased resolution of closely

migrating bands• Potential to lower the units of

enzyme needed for complete restriction

0.1 0.2 0.3 0.35 0.4 0.45 0.5 0.8

E. coli

0.1 0.2 0.3 0.35 0.4 0.45 0.5 0.8

Salmonella

Plug Preparation• Equilibrate melted agarose to

50-54º C• Limit mechanical force

(pipetting) when mixing melted agarose with cell suspensions

• Do not re-use plug agarose more than 3-4 times, since repeated heating results in loss of fluid and increases the agarose concentration

37 ˚ C 56 ˚ C

5 m

in

10 m

in15

min

20 m

in30

min

5 m

in

10 m

in15

min

20 m

in30

min • All protocols that use

lysozyme have 37˚C as the incubation temp

• Found increased lysisefficiency at 56 ˚ C

• Key element in optimizing Listeria PFGE protocol

• May be useful for other Gram positives

Comparison of Lysozyme Incubation Time and Temperatures

SDS in Plug Agarose

• SDS traditionally a part of most PFGE protocols to assist in cell lysis

• Issues with SDS:– Requires excessive washing to

remove from plug following Lysis

– If not removed completely can inhibit restriction

• Found not to be necessary for E.coli, Salmonella, Shigella, Campylobacter, and Vibrio

• Necessary for Listeria

+ - + - + - + + - + - + - + -

Sal

mon

ella

H98

12

E. c

oli

E. c

oli

Shi

gella

dy

sent

eria

Shi

gella

dy

sent

eria

Sal

mon

ella

Litc

hfie

ld

Sal

mon

ella

H98

12

Sal

mon

ella

H

9812

S S

Cell Lysis

• Lyse in 5ml cell lysis buffer (50 mM Tris: 50 mM EDTA, pH 8.0; 1% Sarcosyl; 100 ug/ml Proteinase K per sample) at 54º C with constant and vigorous agitation

• Some organisms lyse better than others– Lysis times may vary– Typically between 1-4 hours

• Plugs typically clear as cells lyse• Incomplete lysis indicated by incomplete

restriction, smearing, and significant fluorescence in the plug slice

Washing of PFGE Plugs• Washes

– Wash in 10-15 ml at 50ºC for 10-15 min with constant agitation

– 2X with sterile clinical laboratory reagent grade water

– 4X with TE buffer (10 mM Tris: 1 mM EDTA, pH 8.0)

• Inadequate washing typically results in incomplete restriction or smearing

• If you suspect plugs were not washed enough– Wash plugs 2X more with TE buffer– Restrict and run another plug slice

Same plugs washed with TE 6X

Plugs washed with TE 4X

Preparing Plug Slices for Restriction Digestion

• Be consistent with the size of plug slices used• Desired width= 2 mm

• Use a sharp tool or device cut plugs with

• Plugs cut too small can easily be damaged and result in band distortions.

• Plug cut too large can result in thick indistinct bands that are difficult to analyze.

Plug Slices 1 mm

Damaged Plug Slices

Restriction Digestion

• Use marked Petri dish, glass slide, or other template to cut plug slices to 2-mm size

• Optimal enzyme will vary between organisms

• Always use the restriction buffer recommended by the vendor

• Always prepare master mixes• Addition of BSA is

recommended even with enzymes that do not require it

Bovine Serum Albumin

BSA is used to maintain enzyme stability during the restriction digestion• Use acetylated or molecular grade for

molecular biology applications• Diluted to a final concentration of 1X

Minimizes enzyme inactivation due to inhibitors. Prevents enzymes from adhering to wall of

reaction tubes BSA can be added to reaction mixtures even

when not recommended by the vendor

Incomplete Restriction

SfiI NotI

Expired SfiI Enzyme

• Described as “shadow” or “ghost” bands on the gel

• May be the result of:– Bad lot of enzyme and/or buffer– Enzyme and/or buffer has gone

bad over time – Poor plug quality – proteinase K

not removed from plug– DNA concentration too high– Enzyme concentration too low– Incorrect incubation

temperature or buffer

10 Units 20 Units 40 Units

• Star activity - a relaxation or alteration of the specificity of a restriction enzyme

• Conditions that can lead to star activity– Prolonged reaction time– Suboptimal buffer or buffer

concentration– High (> 5% v/v) glycerol

concentrations– High concentration of enzyme/µg of

DNA ratio • Often resembles incomplete lysis

AscI ApaI

AscI ApaI

Same plugs re-tested

Overnight Restriction

2 Hour Restriction

Star Activity

Casting Agarose Gel• Level gel form before gel is poured.• Position of comb is important.

• If plugs are loaded into wells, position comb 2-mm above black platform.

• If plug slices are loaded on comb, position comb and plug slices so they are flush against black platform.

• Be sure plug slice is not curved or at an angle. • Do not disturb gel after it is poured and while

still liquid. Remove bubbles or lint with clean pipette tip immediately after gel is poured.

Gel Loading

Allow plug slices to air dry for 5-10 minutes, but do not over dry

Casting of Gel

• Pour melted agarose (equilibrated to ~54°C) slowly from bottom center of gel

• Allow agarose to polymerize at least 30 minutes

• Remove comb• Fill in wells with melted agarose – optional• Place gel into electrophoresis chamber

Electrophoresis• Check milliamperes (mA) at

start of run.• If mA value is high (>165):

– Buffer dilution or formulation problems.

– Water used to prepare 0.5X TBE buffer is of poor quality.

• If mA value is low (<100):– Buffer preparation or dilution

problem.• mA value will increase

during run (~130 mA – 165 mA).

Short Appropriate length

Doublet not resolved

Run Length

• Bottom band (20.5 Kb) of the standard should be 1-1.5 cm from the bottom of the gel

• If the run time is too short– Pattern is compressed– Decreased resolution of closely migrating

bands– Normalization of the pattern may be

compromised

• If the run time is too long– Bottom band of the standard runs off the gel – Unable to perform normalization

• Electrophoresis run time may vary from…• Lab to Lab• Equipment to Equipment• Person to Person• Month to Month

• Critical factors that effect run times• Composition in 0.5X TBE

• Commercial vs. In-house• Buffer pH• Buffer temperature

• Ambient temperature• Length of tubing

• Gel concentration

Run Times

• A Salmonella gel run with E. coli running conditions

• May not notice until performing analysis

• Gel must be rerun to ensure proper resolution of fragments from organism of interest

Wrong Running Condition

Pay attention to distortion bars!!!!!

Wrong Running Conditions

Electrophoresis• Cut tubing connecting electrophoresis

cell and pump to length recommended in the instruction manuals.

• Remove bubbles in the buffer lines.• The circulation and cooling of buffer will be

affected (temperature could rise).• Ice in the line can also affect the circulation

and cooling of buffer.• Eliminate kinks in the tubing.

More About Electrophoresis

• Remove small pieces of agarose from top of gel trap and electrophoresis chamber when buffer is drained.

• If gel trap is not flush with bottom of chamber, small pieces of agarose can circulate through the lines and clog the drain port(s).• May affect buffer circulation.• Replace gel traps with broken “feet” or pegs.

• Rinse chamber and lines with Type I Water to remove residual buffer after runs.

Gel Staining• Stain in Ethidium Bromide

solution for 25-30 minutes– 40 µl of 10 mg/ml stock

solution per 400 ml• Remove stain and de-stain

with ≈500 ml of water• Change water at least

three times every 15-20 minutes.

• As stain staining solution ages background may be observed

Background Due to Inadequate De-Staining

Gel Properly De-Stained

Image Acquisition

•Zoom in to eliminate as much space around the gel as possible

•Do Not take multiple images of the same gel

•Appropriate Image Acquisition

The field of view should be filled as completely as possible with the image of the gel - Zoom

Image Acquisition

• Take measures to ensure the image is in focus• Focus the camera on

the lines of a ruler• May need to minor

adjustments to the field of view once the image is focued

Confirm Focus of CameraNot Focused Focused

Image Acquisition• Avoid over-integration of the image

– Reduces background– Makes closely migrating bands

more distinct• Process

– If available, select “Highlight saturated pixels”

– Saturated areas will appear red– Adjust integration time or the

aperature until all red is removed– Saturation may remain in the wells

• Areas on the gel may appear slightly faint to the naked eye, but will be apparent when analyzing in BioNumerics

Image Acquisition

Increasing Integration Time

• Integration time– Too short = difficulty seeing bottom bands– Too long = Increased background and lose distinction between

closely migrating bands• Take several images and determine which looks best in

BioNumerics

The PulseNet Standard Strain (H9812)

PulseNet saw a need for a “universal” standard which could be used for all organisms

Always test “new” lot of standard plugs with a plug slice from an “old” lot to confirm:

• PFGE pattern is same, including bands intensity• Concentration is ~ the same.• The new standards produce good quality PFGE pattern

Pre-tested H9812 standard plugs can serve as a(+) control for XbaI restriction and focus troubleshooting efforts

• Problem observed in sample lanes but not in standards, likely due to sample associated affect (i.e. sample plug preparation)

• Problem observed in both sample and standard lanes, likely due to step involving sample and standard plugs (i.e. restriction digests)

No Restriction Pattern is Obtained

• Some strains will not restrict with certain enzymes

• Appears as a large band at the top of the gel with no DNA smearing in the lane (red box)

• Recommend – Re-run the isolate one more

time to confirm– If same result is obtained,

upload the information since it is a valid pattern for that strain

E. coli O26, BlnI

Thoiurea-Dependent StrainsWithout Thiourea With Thiourea

Untypeable Result Patterns are Generated• Addition of 50 μM thiourea in 0.5XTBE electrophoresis buffer• Include a positive control on gel

– Restricted PFGE plug slice of “untypeable” strain that “typed” previously when thiourea was used in buffer.

Potential Stopping Points

Lyse plugs overnight (14-16 hours). Refrigerate plugs after first or second wash

with TE; complete TE washes the next day. Restrict plug slices overnight (14-16 h). Not if the enzyme is known to have star

activity (should be stated on product insert) Remove restriction digestion mixture, add TE,

and refrigerate restricted plug slices for up to a week.

Consider All Areas of the PFGE Gel:

Troubleshooting PFGE Gels

Consider all steps of protocol:

• Cell suspension preparation• Preparation of PFGE plugs• Lysis of cells in PFGE plugs• Washing of PFGE plugs• Restriction digestion of DNA• Gel electrophoresis of restricted DNA• Documentation of PFGE gel

S S S

•Determine if anything changed since the last “good” gel.

AcknowledgementsAll PulseNet participants at CDC,

FDA, USDA, and in the State Public Health Laboratories

The findings and conclusions in this presentation are those of the

author and do not necessarily represent the views of the Centers

for Disease Control and Prevention