Let’s start off the new year with some successful transformation experiments! Here are the answers to our most frequently asked questions regarding transformations.
Q: After the incubation period, my source plates have grown more of a lawn of bacteria and no individual colonies are visible. What went wrong?
A: This can happen when the BactoBead is not streaked properly. Once the BactoBead is dissolved with recovery broth, make a primary streak at the top of the plate and rotate the plate 90 degrees. Streak the loop through the primary streak once and create a zig-zag pattern across the clean part of the agar, to make the secondary streak. Continue to follow this streaking pattern until the plate has 4 different streaking sections. This streaking method allows for individual colonies to grow, rather than a lawn of bacteria.
Q: My source plate had little to no growth. What happened?
A: The incubation time could have been too short. Make sure that the plate is being incubated at 37 °C between 16-20 hours. If not enough time is allotted, bacterial growth will be inhibited. If growth on the plate looks limited, even after 16-20 hours, make sure that the incubator is at 37°C by using a thermometer. If you do not have an incubator, or the incubator is not reaching the correct temperature, colonies will form between 24 and 48 hours at room temperature.
Q: How do I add in the antibiotics and additives to the LB agar?
A: After melting the agar, let it cool to 60°C. This is important! If the agar is too hot the antibiotics and additives that are added could degrade, causing issues with bacterial growth and results. Generally, the ampicillin comes as a powder and can be poured into the agar bottle directly. The IPTG comes as a liquid and can be added using a transfer or micropipette!
Q: The transformation didn’t work. The +DNA/AMP and +DNA/AMP/+IPTG plates both have no colonies present. What went wrong?
A: One possibility is that the plates weren’t made properly, where the antibiotics could have been degraded or a component wasn’t added to the plates. Another possibility, which occurs frequently, is that the bacterial cells were not completely resuspended in CaCl2. Make sure that when students are resuspending isolated colonies into the CaCl2, they pipette up and down until no clumps of cells are visible and the cell suspension looks cloudy. This can take some time to accomplish, but is a necessary step! If a vortex is available, it can help speed up the resuspension process. Additionally, ensure that the CaCl2 is ice cold when being used.
Q: My transformation worked, I see colonies on both +DNA/AMP and +DNA/AMP/IPTG plates; however, the colonies on the +DNA/AMP/IPTG plate are not fluorescing when exposed to blue or UV light. What happened?
A: IPTG is responsible for the production of the GFP protein, so if it wasn’t added to the agar properly or if it was degraded, it could cause issues with the fluorescence. Make sure that the entire volume of IPTG was transferred to the agar, and that the agar is cool enough before adding IPTG.
Q: The transformation worked, but very few colonies are present on +DNA/AMP and +DNA/AMP/IPTG plates.
A: There are multiple things that can affect the transformation efficiency. Here are a few possibilities:
-Not enough cells were used for the transformation. Try picking more colonies to resuspend in the CaCl2
-The source plates were too old when the colonies were picked. Make sure to used source plates 18 to 22 hours after they were streaked.
-Either there was too much or too little plasmid DNA added to the cell suspension. Make sure that the correct volume of plasmid DNA is added to resuspended cells.
Try using our enhanced transformation protocol (generally found in the appendix of the literature) to improve transformation efficiency during your experiment!
Please reach out to an Edvotek® Technical Support Specialist at 1-800-EDVOTEK or email us at firstname.lastname@example.org if you have any questions.