2. By: Lynn More - Olympian High School and UCSD Protein Transformation Lab Preview UCSD: BioBridge Program

3. Plate Streaking Techniques

4. Pipette Techniques Squeeze the bulb with two fingers - firmly Insert into the fluid Gently release SOME pressure on the bulb! If you release all pressure the fluid will be sucked up into the bulb rather than to the measurement line. Maintain the pressure Move the pipette to the desired container Squeeze the fluid from the pipette 1 mL 0.5 mL 0.25 mL 0.1 mL 1.00 mL = 1000 µL 0.50 mL = 500 µL 0.25 mL = 250 µL 0.10 mL = 100 µL

5. Micropipette Techniques Press and hold the black “set” button Turn the dial to the desired measurement in µ L Attach the micropipette tip Press the Yellow or Blue Plunger down to the stop Insert the pipette tip into the substance Release the plunger (the proper amount has now been collected) Position the tip over the microcentrifuge tube to dispense, press the plunger to the stop To eject the tip when done, press the plunger all the way down. 1.00 mL = 1000 µL 0.50 mL = 500 µL 0.25 mL = 250 µL 0.10 mL = 100 µL

6. Vortex and Heat Shock To mix the CaCl 2 with the plasmid to neutralize the slightly negative DNA So it will enter the E. coli bacteria. 42˚C for 45 Seconds

7. Get your materials  2 microcentrifuge tubes  4 disposable transfer pipettes tips  3 Inoculating loops  3 cotton swabs  3 LB plates 2 - LB/AMP (red line) 1 - LB, no AMP Store these with the agar on the top Taped together

8. LB/AMP + P4 Table ? LB/AMP - P4 Table ? LB/No AMP - P4 Table ? A+ A- Label your equipment Ready? Set?… Go

9. FP transformation procedure + LB/Amp + - LB/Amp - LB/No amp - Control CaCl 2 + Bacteria + Plasmid (PM1 or PM2) CaCl 2 + Bacteria + Control (TE or dH 2 O) ICE 10 min ICE 2 min 42˚C 45 sec ICE 2 min

10. Materials Checklist  1 microcentrifuge tube of CaCl2 on ice  2 empty microcentrifuge tubes  1 waterproof pen  4 disposable transfer pipette tips  3 Inoculating loops  tape for sealing plates after innoculation  1 LB plate  2 LB/AMP plates  Ice bucket (cup with ice and water)  One tube of plasmid labeled either PM1 or PM2 on ice.  Class or lab station waste containers

11. Recombinant DNA Bacteria cell Bacterial chromosome Bacteria plated on LB agar + antibiotic Only bacteria containing Recombinant DNA grow Collect culture DNA Purification cloning DNA (plasmid) insertion Heat Shock Method Transformation Purification Minimum of 2 days In 37ºC incubator PM1 - blue, green, grape PM2 - Cherry, Tangerine, Banana

12. What’s happening in the petri dish? Ampicillin acts as a __________________ that only allows ___________ bacteria to _____ on the plate Represent ___________________________________________Ampicillin - an antibiotic that inhibits bacterial growth Represent ______________Bacteria growth Represents _________________________________________ LB Agar - a nutrient substrate to encourage growth Represent _________________________________ Genetically transformed bacteria that are: 1. Resistant (or shielded) from the effects of ampicillin 2. Marked with a Fluorescent Protein selection mechanism transformedgrow ______________________Bacteria killed by ampicillin

13. FP transformation procedure