02 - Code Breakers: Using CRISPR to Rewrite Genetics
Transformation Troubleshooting
CRISPR TROUBLESHOOTING GUIDE
PROBLEM:
CAUSE:
ANSWER:
Incubation time too short
Continue to incubate source plate at 37ºC for a total of 18-22 hours.
Poor cell growth on source plate
Incorrect incubation temperature
Use a thermometer to check incubator temperature. Adjust temp. to 37°C if necessary.
Ensure the correct concentration of antibiotic was added to plates - Make sure ReadyPour is cooled to 60°C before adding antibiotic. Make sure ReadyPour is cooled to 60°C before adding antibiotic.
Incorrect concentration of antibiotics in plates Antibiotic is degraded
Satellite colonies seen on experiment plate
Incubate the plates overnight at 37ºC (18-22 hours).
Plates were incubated too long
Plates containing transformants were inverted too soon
Allow cells to fully absorb into the medium before inverting plates.
Colonies appeared smeary on experiment plate
After pouring plates, allow them dry overnight at room temp. Alternatively, warm plates at 37°C for 30 min. before plating cells
Experimental plates too moist
Ensure plasmid DNA was added to tubes.
Plasmid DNA not added to transformation mix
Make sure that pipets are used properly and are properly calibrated.
Incorrect host cells used for experiment
Confirm that correct bacterial strain was used for transformation
No colonies seen on experiment plates
Cells were not properly heat shocked
Ensure that temp. was 42ºC & heat shock step took place for exactly 45 sec.
Incorrect antibiotics
Be certain that the correct antibiotic was used.
Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or pipet up and down to fully resuspend cells). Cell suspension should be cloudy. Fully but gently resuspend cells following centrifugation by slowly pipetting up and down. Ensure CaCl 2 and CCS are cold. Swipe through a dense section of the bacterial culture and use a full match sized loop. Incubate for full times. Keep on ice. Important that source cells grow no longer than 20 hrs. Refrigerate plates after 20 hrs if necessary. Do not use source plates that have been incubated longer than 24 hours (refrigerated or not).
Cells not well resuspended in CaCl 2
Too many injured or non-competent cells.
Not enough cells used for experiment
Source plates were incubated for more than 20 hours
Experimental plates too old
Prepare plate and use shortly after preparation
Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or pipet up and down to fully resuspend cells). Cell suspension should be cloudy. Pre-chill CaCl 2 before adding cells to the CaCl 2 Extend incubation of celll suspension on ice 10-15 min. (should not exceed 30 min. total). This increases the transformation efficiency. Ensure that correct volume of plasmid was added to the transformation tube. If using micropipets, make sure students practice using pipets. Ensure that temperature was 42ºC and that heat shock step took place for no more than 45 seconds.
Low transformation efficiency (only a few colonies seen on experiment plates)
Cells not well resuspended in CaCl 2
CaCl 2 solution not cold enough
Cell solution not cold enough
Too much or too little plasmid DNA added to cell suspension
Cells were not properly heat shocked
Antibiotics were degraded prior to pouring plates
Make sure ReadyPour is cooled to 60°C before adding antibiotic.
Incorrect concentration of antibiotics
Ensure that the correct concentration of antibiotic was used in plates.
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