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

Materials Checklist Students make bags in groups of four: 2 empty microcentrifuge tubes 4 disposable transfer pipette 1 Inoculating loop 6 cotton swabs Teacher will have at lab tables for each group of four: 1 waterproof pen - for labeling 1 LB plate 2 LB/AMP plates Ice bucket (cup with ice and water) 1 microcentrifuge tube of CaCl2 on ice 1 microcentrifuge tube of plasmid labeled either PM1 or PM2 on ice. 2 Lab station waste containers - one with 10% bleach, other empty Tape for sealing plates after inoculation Timer

1. Untape petri dishes and put them on the placemat where they go. Save the tape for the end! 2. Label equipment 3. Perform Transformation Lab 4. Incubate your plates 5. Turn in your group’s “What Do You Know” sheet

Labeling Equipment + and Table letter - and Table letter LB/Amp + Period & Table LB/Amp - Period & Table LB/ no Amp - Period & Table

Equipment 0.5 mL Transfer Pipette 0.25 mL 0.1 mL Microcentrifuge Tube

FP Transformation procedure Suspend bacterial colonies in CaCl2 Add plasmid DNA Incubate on ice for 10 minutes Work on you’re the “What Do You Know?” info

FP Transformation procedure Suspend bacterial colonies in CaCl2 Add plasmid DNA Heat-shock: • ice for 10 minutes • 42°C for 45 seconds • back on ice again for 2 min Plate out bacteria Read & Follow your lab instructions!

Transformation Flow Chart + - CaCl2 + Bacteria Plasmid (PM1 or PM2) CaCl2 + Bacteria Incubate on ice for 10 minutes Heat Shock: Ice – Heat - Ice LB/Amp + LB/Amp - LB/No Amp - Experimental Plate: Colonies of bacteria With plasmids Control Plate: LB/Amp-: Testing effectiveness Of Ampicillin And contamination Control Plate: LB/No Amp-: “Lawn” testing viability of the bacteria and the nutrient Purpose of Each plate

What is in the plate to start with? What do you expect to see? What do you know? Plate What is in the plate to start with? What do you expect to see? Experimental Plate: LB/Amp + Control Plate: LB/Amp - Control Plate: LB/No Amp -

What is in the plate to start with? What do you expect to see? What do you know? (KEY) Plate What is in the plate to start with? What do you expect to see? • Luria Broth (Nutrient/Food) • Ampicillin (antibiotic) (tainted food) • transformed bacteria (bacteria + the plasmid) Glowing bacteria survive (regular bacteria die off) transformed bacteria Colored bacteria • regular bacteria (bacteria minus plasmid) No growth at all (ampicillin kills off the regualr bacteria like it’s supposed to) • Regular food, Food is NOT tainted A lot of growth (lawn) (give regular bacteria good food and it’ll GROW!) Experimental Plate: LB/Amp + Control Plate: LB/Amp - Control Plate: LB/No Amp -