Frequent Difficulties With PFGE (Troubleshooting Tips) 6 th PulseNet Latin America Meeting Buenos Aires, Argentina June 26 th 2008 Efrain M. Ribot, Ph.D. PulseNet Methods Development Laboratory Centers for Disease Control and Prevention Atlanta, 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 for reagents is the best possible. Change filters in water system on regular basis. Use sterile ultrapure (Type I or Reagent Grade) water for all reagents. Non-sterile ultrapure water can be used for: Buffer used to make gels. Electrophoresis running buffer De-staining gels
Non-selective Media Culture Growth Selective Media 0.35 0.45 0.6 0.35 0.45 0.6 Cell suspension 18 hrs 48 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 ready to limit exposure of the organism to oxygen
Cell Suspension Preparation Mix cell suspension well, but DO NOT VORTEX Check concentration value at least twice More (higher concentration of cells) is not always better.
Plug Preparation Equilibrate melted agarose to 50-54 54º C Limit mechanical force (pipetting)) when mixing melted agarose with cell suspensions Do not re-use plug agarose more than 3-43 4 times, since repeated heating results in loss of fluid and increases the agarose concentration
Lysis Make master mixes for Cell Lysis Buffer and Proteinase K Lyse plugs in a shaking water bath or incubator at 54º C. Use no less than 5ml of lysis buffer per isolate. Follow lysis time recommended in the protocol for the organism being tested.
Plug Washing: To remove Impurities that may inhibit restriction digestion Inadequate Washing Washing two additional times Perform washes in no less 10-15 15 ml to allow impurities 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 for Restriction Digestion Be consistent with the size of plug slices used 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
Preparing Plug Slices for Restriction Digestion H9812 Double Digest -Enzyme not inactivated prior to gel loading Always use master mixes when preparing enzyme mixtures Routinely check water bath temperatures Incubate with 0.5 X TBE to inactivate enzyme
Restriction of DNA Incomplete Complete Incomplete restriction (shadow or ghost bands) is often associated with impurities not removed during washing (i.e. proteinase K) Incomplete restriction could be related to specific lots of enzymes or impurities in water used to prepare restriction mixture
More on Incomplete Restriction Other 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 standard strain or other sample that previously gave a 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 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.
Loading plugs
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).
Run Times Bottom band (20.5 Kb) of the standard should be 1-1.51 1.5 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 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!!!!!
Electrophoresis 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. Cut tubing connecting electrophoresis cell and pump to length recommended in the instruction manuals. 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. feet. Rinse chamber and lines with Type I Water to remove 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 every 15-20 minutes.
Image Acquisition Take measures to ensure the image is in focus Focus the camera on the lines of a ruler
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 File Conversion for GelDoc EQ and XR (Quantity One Software version 4.5.0 or higher). Some value that when you hit enter converts the file size to ~300 Kb
Confirm Focus of Camera Not Focused Focused
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.
What are Untypeable Strains? Some isolates only give a smear on gel and are considered to be untypeable by PFGE. Not able to determine if related isolates have the same or different PFGE patterns. No information for epidemiological studies. First observed at CDC with E. coli O157:H7 Phage Type 31 strains
Un-typeable Strains Thiourea Addition of 50 µm thiourea in the electrophoresis buffer (0.5X TBE) Use only when needed (with known or suspected untypable strains) 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 with PFGE. Other bacteria include: Clostridia Vibrio species Pseudomonas aeruginosa Klebsiella
Precautions Adding thiourea to the running buffer will not solve 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 thiourea was used in buffer. Long term effects on the electrophoresis chamber, tubing and connections are not known.
Pre-test PFGE Plugs of H9812, the PulseNet Standard Strain Always test new lot of standard plugs with old lot to confirm: PFGE pattern is same, including intensity of bands. The new standards produce good quality PFGE pattern Pre-tested H9812 standard plugs can serve as a (+) control for XbaI restrictions and focus troubleshooting efforts. If problem observed in sample lanes but not in standards, is was due to something that only effects the sample (i.e. sample plug preparation) If problem is observed in both sample and standard lanes, it is due to a step involving sample and standard plugs (i.e. restriction ion digests)
Electrophoresis Chamber Contamination Electrophoresis unit was probably contaminated with bacteria or fungi. Pseudomonas species isolated from CDC unit. To eliminate contamination: Circulate 2 liters of a 5%-10% solution of bleach through electrophoresis chamber and tubing for 30 minutes. Do not turn on chiller. Drain bleach solution from chamber. Circulate 2 liters of Type 1 water for 15-30 minutes through chamber and tubing (2X). Run gel with λ ladder, yeast chromosomes, and/or PFGE plugs tested previously
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