THE BIOTECHNOLOGY
EDUCATION COMPANY
EDUCATION COMPANY
Troubleshooting Guide:
Transformation
| PROBLEM: | CAUSE: | ANSWER: |
|---|---|---|
| Poor cell growth on source plate | Incubation time too short | Continue to incubate source plate at 37˚C for a total of 16-20 hours |
| Antibiotic added to source plate plate | When pouring plates, be sure to add antibiotics & additives at the correct step | |
| Incorrect incubation temperature | Use a thermometer to check incubator temperature. Adjust temp. to 37˚C if necessary | |
| Satellite colonies seen on transformation plate | Incorrect concentration of antibiotics in plates | Ensure the correct concentration of antibiotic was added to plates. Make sure ReadyPour is cooled to 60˚C before adding antibiotic |
| Antibiotic is degraded | Make sure ReadyPour is cooled to 60˚C before adding antibiotic | |
| Plates were incubated for too long | Incubate the plates overnight at 37˚C (16-18 hours) | |
| Colonies appeared smeary on transformation plate | Plates containing transformants were inverted too soon | Allow cell suspension to fully be absorbed into the medium before inverting plates |
| Experimental plates too moist | After 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 plates | Plasmid DNA not added to transformation mix | Ensure 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 transformation | Confirm that correct bacterial strain was uesd for transformation | |
| Cells were not properly heat shocked | Ensure that temperature was 42˚C & heat shock step took place for no more than 90 seconds | |
| Incorrect antibiotics | Be certain that the correct antibiotic was used | |
| Low transformation efficiency | Not enough cells used for transformation | Pick more colonies from source plate (15 colonies @ 1-2 mm width per 500µl CaCl2) |
| Source plates were incubated for more than 20 hours | Important 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 old | Prepare transformation plate and use shortly after preparation | |
| Cells not well resuspended in CaCl2 | Completely 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 enough | Pre-chill CaCl2 before adding cells to the CaCl2 | |
| Cell solution not cold enough | Extend 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 suspension | Ensure 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 shocked | Ensure 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 plates | Make sure ReadyPour is cooled to 60˚C before adding antibiotic | |
| Incorrect concentration of antibiotics in plates | Ensure that the correct concentration of antibiotic was used |