• Home
  • transformation troubleshooting guide

Troubleshooting Guide:

Transformation

PROBLEM:CAUSE:ANSWER:
Poor cell growth on source plateIncubation time too shortContinue to incubate source plate at 37˚C for a total of 16-20 hours
Antibiotic added to source plate plateWhen pouring plates, be sure to add antibiotics & additives at the correct step
Incorrect incubation temperatureUse a thermometer to check incubator temperature. Adjust temp. to 37˚C if necessary
Satellite colonies seen on transformation plateIncorrect concentration of antibiotics in platesEnsure the correct concentration of antibiotic was added to plates. Make sure ReadyPour is cooled to 60˚C before adding antibiotic
Antibiotic is degradedMake sure ReadyPour is cooled to 60˚C before adding antibiotic
Plates were incubated for too longIncubate the plates overnight at 37˚C (16-18 hours)
Colonies appeared smeary on transformation platePlates containing transformants were inverted too soonAllow cell suspension to fully be absorbed into the medium before inverting plates
Experimental plates too moistAfter pouring plates, allow them to dry overnight at room temperature. Alternatively, warm plates at 37˚C for 30 minutes before plating cells
No colonies seen on transformation platesPlasmid DNA not added to transformation mixEnsure plasmid DNA was added to transformation tube
Make sure that pipets are used properly. If using micropipets, make sure students practice using pipets
Incorrect host cells used for transformationConfirm that correct bacterial strain was uesd for transformation
Cells were not properly heat shockedEnsure that temperature was 42˚C & heat shock step took place for no more than 90 seconds
Incorrect antibioticsBe certain that the correct antibiotic was used
Low transformation efficiencyNot enough cells used for transformationPick more colonies from source plate (15 colonies @ 1-2 mm width per 500µl CaCl2)
Source plates were incubated for more than 20 hoursImportant that source cells grow no longer than 20 hours. Refrigerate plates after 20 hours if necessary. Do not use source plates that have been incubated longer than 24 hours, refrigerated or not)
Experimental plates too oldPrepare transformation plate and use shortly after preparation
Cells not well resuspended in CaCl2Completely resuspend the cells in the CaCl2, leaving no cell cllulmps (vortex or mix vigorously to full resuspend cells). Cell suspension should be cloudy
CaCl2 solution not cold enoughPre-chill CaCl2 before adding cells to the CaCl2
Cell solution not cold enoughExtend incubation of cells in CaCl2 + DNA on ice (extra 10-15 minutes but not more than 30 minutes). This allows more DNA to adhere to outside of bacterial cell
Too much or too little plasmid DNA added to cell suspensionEnsure that correct volume of plasmid was added to the transformation tube. If using micropipets, make sure students practice using pipets
Cells were not properly heat shockedEnsure that temperature was 42˚C and that the heat shock step took place for no more than 90 seconds
Antibiotics were degraded prior to pouring platesMake sure ReadyPour is cooled to 60˚C before adding antibiotic
Incorrect concentration of antibiotics in platesEnsure that the correct concentration of antibiotic was used

Printer-Friendly PDF