Color Your Classroom: Engaging Students with Bacteria and Bio-Art
Experiment Procedure
Transformation of E. coli with Blue, Pink, Purple Chromogenic Proteins
500 µL CaCl 2
Transfer 5 colonies
For best results, make sure that the cells are completely resuspended.
E.coli source plate
42°C
250 µL
ADD: 10 µL Plasmid DNA DO NOT ADD TO THE “-DNA” TUBE!
37°C
-DNA +Amp
Make sure to keep the actual labels small!
-DNA
250 µL Recovery Broth
+DNA +Amp +IPTG
+DNA +Amp
1. LABEL one microcentrifuge tube with “+DNA” and a second microcentrifuge tube with “-DNA”. 2. TRANSFER 500 µL ice-cold CaCl 2 solution into the ”– DNA” tube using a sterile 1 mL pipet. 3. Using a toothpick, TRANSFER 5 well-isolated colonies (each colony should be approx. 1-1.5 mm in size) from the E. coli source plate to the “-DNA” tube. 4. RESUSPEND the bacterial cells in the CaCl 2 solution by pipetting up and down until no clumps of cells are visible and the cell suspension looks cloudy. 5. TRANSFER 250 µL of the cell suspension to the tube labeled “+ DNA”. PLACE tubes on ice. 6. ADD 10 µL of the Plasmid DNA to the tube labeled “+ DNA”. DO NOT add the plasmid to the “-DNA” tube. 7. Gently MIX the samples by flicking the tubes. INCUBATE the tubes on ice for 10 minutes. 8. PLACE the transformation tubes in a 42°C water bath for 45 seconds. 9. Immediately RETURN the tubes to the ice bucket and INCUBATE for two minutes. 10. TRANSFER 250 µL of Recovery Broth to each tube using a sterile 1 mL pipet. Gently MIX by flicking the tube. 11. INCUBATE the cells for 10 minutes in a 37°C water bath. 12. While the cells are recovering, LABEL the bottom of four agar plates as indicated below. • -DNA (plate with no stripe) • -DNA/+Amp (plate with one stripe) • +DNA/+Amp (plate with one stripe) +DNA/+Amp/+IPTG (plate with two stripes)
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