Frequent Difficulties With PFGE - Inocuidade de Alimentos

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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 recommen<strong>de</strong>d in the protocolfor the organism being tested.
Page 1 and 2: Frequent Difficulties With PFGE(Tro
Page 3 and 4: Water Quality• Make sure the qual
Page 5: Cell Suspension Preparation• Mix
Page 9 and 10: Preparing Plug Slices forRestrictio
Page 11 and 12: Restriction of DNAIncompleteComplet
Page 13 and 14: Casting Agarose Gel• Level gel fo
Page 15 and 16: Casting of Gel• Pour melted agaro
Page 17 and 18: Run Times• Bottom band (20.5 Kb)
Page 19 and 20: Wrong Running Condition• A Salmon
Page 21 and 22: Electrophoresis• Remove bubbles i
Page 23 and 24: Gel Staining• Stain in Ethidium B
Page 25 and 26: Image Acquisition• Avoid over-int
Page 27 and 28: Confirm Focus of CameraNot FocusedF
Page 29 and 30: What are “Untypeable” Strains?
Page 31 and 32: Examples of “Untypeable” Strain
Page 33 and 34: Pre-test PFGE Plugs of H9812, thePu
Page 35: Send Your TroubleshootingQuestions

Frequent Difficulties With PFGE - Inocuidade de Alimentos

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