Frequent Difficulties With PFGE - Inocuidade de Alimentos

Frequent Difficulties With PFGE(Troubleshooting Tips)6 th PulseNet Latin America MeetingBuenos Aires, ArgentinaJune 26 th 2008Efrain M. Ribot, Ph.D.PulseNet Methods Development LaboratoryCenters for Disease Control and PreventionAtlanta, GA

Troubleshooting PFGE Gels• Consider all steps in protocol:• Preparation of cell suspensions• Preparation of PFGE plugs• Lysis of cells in PFGE plugs• Washing of PFGE plugs• Restriction digestion of DNA• Electrophoresis of restricted DNA• Documentation of PFGE gel• Equipment performance• Changes in laboratory practices, vendors,reagent preparers, etc.

Water Quality• Make sure the quality of water used forreagents is the best possible.• Change filters in water system on regular basis.• Use sterile ultrapure (Type I or ReagentGrade) water for all reagents.• Non-sterileultrapure water can be used for:• Buffer used to make gels.• Electrophoresis running buffer• De-staining gels

Non-selectiveMediaCulture GrowthSelectiveMedia0.35 0.45 0.6 0.35 0.45 0.6Cellsuspension18 hrs48 hrs• Grow cultures on non-selective media• Plugs should be made from fresh cultures– 14-18 18 hours old• Prepare Campylobacter plugs as soon as culture is readyto limit exposure of the organism to oxygen

Cell Suspension Preparation• Mix cell suspension well,but DO NOT VORTEX• Check concentrationvalue at least twice• More (higherconcentration of cells) isnot always better.

Plug Preparation• Equilibrate melted agarose to50-5454º C• Limit mechanical force(pipetting)) when mixingmelted agarose with cellsuspensions• Do not re-use plug agarosemore than 3-434 times, sincerepeated heating results inloss of fluid and increasesthe agarose concentration

Lysis• Make “master mixes” for Cell Lysis Buffer andProteinase K• Lyse plugs in a shaking water bath or incubatorat 54º C.• Use no less than 5ml of lysis buffer per isolate.• Follow lysis time recommended in the protocolfor the organism being tested.

Plug Washing:To remove Impurities that may inhibitrestriction digestionInadequate WashingWashing two additional times• Perform washes in no less 10-15 15 ml to allowimpurities to diffuse out.• At least 2 water washes and 4 TE washes• Wash at 50ºC in a shaking water bath or incubator

Preparing Plug Slices forRestriction Digestion• Be consistent with thesize of plug slices used• Use a sharp tool or devicecut plugs with• Plugs cut too small caneasily be damaged andresult in band distortions.• Plug cut too large canresult in thick indistinctbands that are difficult toanalyze.PlugSlices1 mmDamagedPlug Slices

Preparing Plug Slices forRestriction DigestionH9812 Double Digest -Enzyme notinactivated prior to gel loading• Always use mastermixes when preparingenzyme mixtures• Routinely check waterbath temperatures• Incubate with 0.5 XTBE to inactivateenzyme

Restriction of DNAIncompleteComplete• Incomplete restriction(shadow or ghost bands) isoften associated withimpurities not removedduring washing (i.e.proteinase K)• Incomplete restriction couldbe related to specific lots ofenzymes or impurities inwater used to preparerestriction mixture

More on Incomplete RestrictionOther reasons for incomplete restriction:• Poor plug quality – SDS, Proteinase K,Sarcosine were not removed from plug.• Concentration of DNA was too high.• Concentration of enzyme was too low.• Wrong incubation temperature or buffer.• Plug slice was not under restriction mixture.• Bad lot of enzyme and/or buffer.• Restrict PFGE plug slice of the standardstrain or other sample that previously gavea clean pattern to check.

Casting Agarose Gel• Level gel form before gel is poured.• Position of comb is important.• If plugs are loaded into wells, position comb2-mm above black platform.• If plug slices are loaded on comb, positioncomb and plug slices so they are flushagainst black platform.• Be sure plug slice is not curved or at an angle.• Do not disturb gel after it is poured and whilestill liquid. Remove bubbles or lint with cleanpipette tip immediately after gel is poured.

Loading plugs

Casting of Gel• Pour melted agarose (equilibrated to~54°C) slowly from bottom center of gel• Allow agarose to polymerize at least 30minutes• Remove comb• Fill in wells with melted agarose – optional• Place gel into electrophoresis chamber

Electrophoresis• Check milliamperes (mA)) atstart of run.• If mA value is high (>165):– Buffer dilution or formulationproblems.– Water used to prepare 0.5XTBE buffer is of poor quality.• If mA value is low (

Run Times• Bottom band (20.5 Kb) of the standard shouldbe 1-1.511.5 mm 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

Run Times• 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

Wrong Running Condition• A Salmonella gel run withE. coli running conditions• May not notice untilperforming analysis• Gel must be rerun toensure proper resolutionof fragments fromorganism of interest

Wrong Running ConditionPay attention to distortion bars!!!!!

Electrophoresis• Remove bubbles in the buffer lines.• The circulation and cooling of buffer will beaffected (temperature could rise).• Ice in the line can also affect the circulationand cooling of buffer.• Cut tubing connecting electrophoresiscell and pump to length recommendedin the instruction manuals.• Eliminate kinks in the tubing.

More About Electrophoresis• Remove small pieces of agarose from top ofgel trap and electrophoresis chamber whenbuffer is drained.• If gel trap is not flush with bottom of chamber,small pieces of agarose can circulate throughthe lines and clog the drain port(s).• May affect buffer circulation.• Replace gel traps with broken “feet. feet.”• Rinse chamber and lines with Type I Water toremove residual buffer after runs.

Gel Staining• Stain in Ethidium Bromide solution• 40 µl l of 10 mg/ml stock solution per 400 ml• Stain gel on rocker for 25-30 minutes• Remove stain and de-stain with ≈500 ml• Change water at least three times every15-20 minutes.

Image Acquisition• Take measuresto ensure theimage is in focus• Focus thecamera on thelines of a ruler

Image Acquisition• Avoid over-integration of the image– Reduces background– Makes closely migrating bandsmore distinct• Process– If available, select “Highlightsaturated pixels”– Saturated areas will appear red– Adjust integration time or theaperature until all red is removed– Saturation may remain in the wells• Areas on the gel may appearslightly faint to the naked eye, butwill be apparent when analyzing inBioNumerics

Image AcquisitionFile Conversion for GelDoc EQ and XR(Quantity One Software – version 4.5.0 or higher).Some value thatwhen you hit“enter” convertsthe file size to~300 Kb

Confirm Focus of CameraNot FocusedFocused

Bovine Serum Albumin• BSA is used to maintain enzyme stabilityduring the restriction digestion• Use acetylated or molecular grade formolecular biology applications.• Diluted to a final concentration of 1X• Minimizes enzyme inactivation due toinhibitors.• Prevents enzymes from adhering to wall ofreaction tubes.• BSA can be added to reaction mixtureseven when not recommended by thevendor.

What are “Untypeable” Strains?• Some isolates only give a “smear” on gel andare considered to be “untypeable” by PFGE.• Not able to determine if related isolates havethe same or different PFGE patterns.• No information for epidemiological studies.• First observed at CDC with E. coli O157:H7Phage Type 31 strains

Un-typeableStrainsThiourea• Addition of 50 µM thiourea in the electrophoresis buffer(0.5X TBE)• Use only when needed (with known or suspected untypablestrains)• Safety concerns• Not a substitute for poor quality plugs

Examples of “Untypeable” Strains• O157:H7 Phage Type 31 strains• Some serotypes of Salmonella - Ohio, Panama,Saintpaul, , Kentucky, and Livingston,• Some strains of various Salmonella serotypes.• Newport, Miami, and others that usually type withPFGE.• Other bacteria include:• Clostridia• Vibrio species• Pseudomonas aeruginosa• Klebsiella

Precautions• Adding thiourea to the running buffer will notsolve the problems of poor plug preparation.• Do not use on routine gels unless necessary.• Include a positive control on gel.• Restricted PFGE plug slice of “untypeable”strain that “typed” previously when thioureawas used in buffer.• Long term effects on the electrophoresischamber, tubing and connections are notknown.

Pre-test PFGE Plugs of H9812, thePulseNet Standard Strain• Always test “new” lot of standard plugs with “old” lot toconfirm:• PFGE pattern is same, including intensity of bands.• The new standards produce good quality PFGEpattern• Pre-tested H9812 standard plugs can serve as a(+) control for XbaI restrictions and focus troubleshootingefforts.• If problem observed in sample lanes but not in standards, is wasdue to something that only effects the sample (i.e. sample plugpreparation)• If problem is observed in both sample and standard lanes, it isdue to a step involving sample and standard plugs (i.e. restrictioniondigests)

Electrophoresis ChamberContamination• Electrophoresis unit was probablycontaminated with bacteria or fungi.• Pseudomonas species isolated fromCDC unit.• To eliminate contamination:• Circulate 2 liters of a 5%-10%solution of bleach throughelectrophoresis chamber and tubingfor 30 minutes. Do not turn on chiller.• Drain bleach solution from chamber.• Circulate 2 liters of Type 1 water for15-30 minutes through chamber andtubing (2X).• Run gel with λ ladder, yeastchromosomes, and/or PFGE plugstested previously

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