Color Your Classroom: Engaging Students with Bacteria and Bio-Art
Experiment Procedure, continued
Transformation of E. coli with Blue, Pink, Purple Chromogenic Proteins
250 µL
250 µL
250 µL
250 µL
+DNA/+Amp/ +IPTG
-DNA/+Amp
-DNA
+DNA/+Amp
37°C
Cover & Wait
13. After the recovery period, REMOVE the tubes from the water bath and place them on the lab bench. 14. Using a sterile 1 mL pipet, TRANSFER 250 µL recovered cells from the tube labeled “ –DNA “ to the middle of the -DNA and -DNA/+Amp plates. 15. Using a new sterile 1 mL pipet, TRANSFER 250 µL recovered cells from the tube labeled “ +DNA “ to the middle of the +DNA/+Amp and +DNA/+Amp/+IPTG plates. 16. SPREAD the cells over the entire plate using an inoculating loop. Use one sterile loop to spread both -DNA samples. Change to a fresh loop before spreading the +DNA samples. Make sure the cells have been spread over the entire surface of the plates. COVER the plates and WAIT five minutes for the cell suspension to be absorbed by the agar. 17. STACK the plates on top of one another and TAPE them together. LABEL the plates with your initials or group number. PLACE the plates in the inverted position (agar side on top) in a 37°C bacterial incubation oven for overnight incubation (24 hours). If you do not have an incubator, colonies will form at room temperature in approximately 24 - 48 hours. 18. VISUALIZE the transformation and control plates and RECORD the following: • The number of colonies on the plate. • The color of the bacteria.
Experiment Summary: E. coli from the source plate are resuspended in an ice-cold CaCl2 solution. Plasmid DNA is added to half of the cells be- fore they are “heat shocked” in a 42°C water bath. The heat shock step facilitates the entry of DNA into the bacte- rial cells. Recovery Broth is added to the cell suspension, and the bacteria are allowed to recover for 10 minutes at 37°C. This recovery period allows the bacteria to repair their cell walls and to express the antibiotic resistance gene. Lastly, the transformed E. coli are plated on LB plates and allowed to grow at 37°C overnight.
NOTE for Step 17: It may take longer for the cells to absorb into the medium. Do not invert plates if cells have not completely been absorbed.
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