Essential Steps for Prepping AP Lab 6B: Bacterial Transformation.
Overview: There are 6 essential steps for preparing and performing the BioRad “pGLO” transformation with students. This document briefly outlines the prep guide and references excellent resource material in the 66 page prep guide from Bio-Rad.
Step I: Preparing Plates (3-7 Days)
Follow the protocol described on pp. 13-14, but keep the following in mind:
A. Labeling plates. i. Leave the bottom of 16 plates empty. These will be your LB Plates ii. Use a Sharpie to draw one 1cm “dash” on the bottom of sixteen other plates. These will be your LB/amp plates. iii. Use a Sharpie to draw two 2cm “dashes” on the bottom of the remaining 8 plates. These will be your LB/amp/ara plates
B. Mixing reagents. Follow the protocol on p. 9. (500 ml distilled H2O (dH2O) into a
1L flask, add LB agar powder, microwave until dissolved.
C. (If your microwave isn’t big enough for a 1L flask, place 250 ml dH2O into a 500 m flask and add ½ the LB agar powder. Do this twice. microwave until dissolved.
D. Create stacks of four plates each and pour dissolved LB agar (plus antibiotics and extra sugars) as described on p. 11. See Step II for preparation of antibiotics and extra sugars.
E.
Why these plates…
Plate Contents
LB (each student group will have one)
LBamp (each group will have two )
LB amp/ara
(each group will have one)
Code
No mark
1 mark
2 marks
Purpose of Plate
Positive control: indicates that the “heat shock” procedure doesn’t kill E. coli. Serves as a comparison between for nontransfomed cells and transformed cells
Plate one: negative control: indicates that antibiotic does kill non-transformed bacteia. Plate Two: evidence that transformation occurred
Contains transformed cells that will glow under UV light. The arabinose is an inducer that interacts with the promoter (a gene). The promoter “switches on” other genes, in this case the gene for GFP.
F. Store plates overnight (O/N) at room temp (RT) bottom-side down.
G. Store plates for 3-7 days (wrapped in cellophane/shrink wrap) at RT bottom-side up
Step II. Mixing antibiotics and sugars (follow diagram on p. 14)
A. Add 3 ml CaCl
2
to ampiciliin, flick with your finger, let sit for 5 minutes
B. Add 3 ml CaCl
2
to arabinose, flick with finger, mix every minute for 10-15 minutes.
C. Place these mixtures in the fridge while you dissolve your LB agar in the microwave.
D. Follow the procedure on p. 13 and p. 15 for pouring LB and then adding necessary reagents (ampicillin first, arabinose second) and continuing to pour plates

Step III. Rehydrate E. coli and streaking plates
A. Following the procedure on page 16. Add 250 ul of CaCl
2
to the vial with dry
(lyophilized E. coli). You probably won’t see any change in the solution, but let it sit for 5 minutes, trust the E. coli are suspended in solution and steak plates.
B. Streak plates using the protocol described on pp. 16-17. Dip a loop in the vial of E. coli, hold loop at 45° angle to plate, and “sketch” the loop across the plate with your dominant hand while turning the plate with your other hand.
The objective is to distribute the E. coli evenly across the plate.
C. IF THIS WORKS YOU WILL HAVE SINGLE COLONIES OF E. Coli.
D. Following streaking, invert the plates and place in a 37° incubator. Check your plates the next day and monitor them over two days.
E.
IF YOU DO NOT GET SINGLE COLONIES OF E. coli, (smooth, “glossy” round colonies with a dis tinct odor) STOP. DON’T WASTE CLASS TIME
TRYING TO TRANSFORM SOMETHING OTHER THAN E. coli. White,
“fluffy” or rough-edged colonies are likely fungus. We don’t have the technology in the AP Bio lab to transform eukaytotes.
F. Protocol says DO NOT REFRIGERATE. If you refrigerate plates, you will get better (2-10x better) results. I suggest refrigerating two of the plates and leaving two in the incubator. Let students choose starter colonies and then compare results.
Step IV. If you get E. coli, then set up student groups of plates. They should be properly coded, wrapped in skrink wrap and stored at RT for students to pick up.
Step V. Morning of the Lab rehydrate the pGLO plasmid. Transfer 250 ul of CaCl to the clear vial containing lyophilized (freeze-dried) pGLO plasmid. Leave this in the fridge or on ice until class time.
Step VI. Aliquot solutions and set up student racks (image to follow).
Make sure student groups have access to a starter plate of E. coli, a 42°C water bath and a 37° incubator. They will also need a Sharpie and labeling tape (if available). Each group should have their own small cooler or cup (I like a 20-oz. Styrofoam cup filled with crushed ice).
Step VII. Students should follow the protocol printed on the quick guide (pp. 18-19) of the Bio-Rad protocol. Here are some hints:
A. On step 6, it needs to be at least 10 mintutes . Longer is okay
B. On step 8, the heat shock transfer needs to be FAST, FAST FAST, take cooler to water bath and perform at the water bath. Don’t let students walk around with E. coli out of the cooler or water bath.
C. On step 9, the 10 minute incubation can occur at 37° (in an incubator) this will increase transformation efficiency
Remember the over all objective: If one student group is successful transforming bacteria, has growth on the LB amp plate and can induce glowing under UV light on the
LB amp/ara plate, then you have a successful lab.