1 26 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN Bacterial Transformation Protocol 2 Group # Role in Group Materials Reader Timer Technician Student Name Materials checklist (1) ScienceBridge Transformation Protocol (1) Agar plate containing LB/No Amp (2) Agar plate containing LB/AMP plates (red mark on side) (1) Sterile inoculating loop (4) Plastic transfer pipettes (2) Clear 1.5 ml micro tubes (2) Cotton swabs (1) Sharpie Lab procedure NOTE: When labeling plates, write on the BOTTOM of the plate (not the lid) and keep writing small and close to the edges! (1) Piece of tape for sealing plates after inoculation (1) Waste container (1) Styrofoam cup with ice ON ICE (1) Blue 1.5mL tube of CaCl 2 (1) Colored 1.5mL tube of plasmid DNA labeled either PM1 or PM2 Shared materials for class Hot water bath at 42 º C (4 per class) Starter bacterial plate 1. Label plates all three plates with the plate number, date, class period and your group number or initials as listed below: Plate # Label LB / Amp + LB / Amp - LB / (no Amp) - 1 LB/Amp (+) (red line) 2 LB/Amp ( ) (red line) #1 #2 #3 3 LB/No Amp ( ) (no red line) 2. Close the caps on two microtubes and label each cap with a sharpie: one tube with a +, other tube with a + -
2 PROTOCOL Using a plastic transfer pipette transfer 0.5 ml of CaCl2 to both tubes, close and place them on ice for at least 2 minutes. Discard the pipette in the waste container. CaCl ml Using a sterile loop, gently collect ONE colony ( dot ) of bacteria from the top of the class starter plate. Transfer the collected colony to one tube of CaCl 2. Swirl and twist the loop to make sure all the bacteria mix with the CaCl 2 solution. Mix the contents by inverting the tube or flicking the bottom of the tube. The solution should look cloudy with no chunks. 5. Transfer ONE additional colony to the second tube and repeat instructions in #4. Place the tubes back on ice. 6. Using a clean plastic pipette, add all of the plasmid mix solution (labeled either PM1 or PM2 ) into the positive (+) tube with CaCl 2 BE SURE THAT THE PLASMID IS ONLY TRANSFERRED TO THE + TUBE. Mix. Discard the used pipette. DNA (plasmid) + 7. Incubate both tubes on ice for 10 minutes. Make sure the tubes are immersed in the ice. 10 minutes
3 28 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN 8. The timer and one other group member will Check the water bath temperature to ensure it is at 42 º C. Hold the tubes in the hot water for exactly 45 seconds. Make sure that the tubes are in contact with the hot water Immediately return the tubes to the ice for 2 minutes. 42 o 45 seconds 2 minutes 9. Invert your tubes gently to mix. Using a new pipette, transfer 0.25 ml of the cell mixture from the (-) negative tube to the LB/No Amp (-) agar plate. Spread the mixture around the plate gently with a clean cotton swab ml LB / (No Amp) - #3 10. With the same pipette, transfer 0.25 ml of the cell mixture from the (-) negative tube to the LB/Amp (-) agar plate. Spread the mixture around the plate gently with the same cotton swab. Make sure to completely finish the two - (negative) plates before spreading the cells on the + (positive) plate ml LB /Amp - #2 11. With a new clean pipette, transfer 0.25 ml of the cell mixture from the (+) positive tube to the LB/Amp (+) plate. Spread the mixture around the plate gently with a clean cotton swab ml LB /Amp + #1 37 o 12. Stack your plates, tape them together, put plates UPSIDE DOWN to grow overnight in 37 º C incubator.
4 PROTOCOL 2 Bacterial Transformation Using Fluorescent Protein Protocol 2 29 Post lab questions Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line) Plasmid Present? YES NO YES NO YES NO Ampicillin present in plate? YES NO YES NO YES NO Actual Plate Results (Drawing) Actual Plate Results (Description) 1. Estimate the number of fluorescent colonies that grew on your plates. 2. Which plasmid mix did you have and which three colors of fluorescent bacteria did you observe? (Yes, three colors are present! Look again!)
5 30 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN Analysis / discussion of data 1. What made it possible for the colonies to be different colors? 2. Describe how the plasmid was able to enter the cell. 3. Suppose that a group did not get any transformed colonies. What possible errors could have caused this? Conclusion / summary (revisit hypothesis)
6 APPENDIX 1 31 Transformation Protocol Flow Chart Positive Tube (+) Negative Tube (-) 500 µl CaCl 2 ON ICE! 500 µl CaCl 2 ON ICE! (1) Bacteria Colony Vortex or Shake to Mix (1) Bacteria Colony Vortex or Shake to Mix 100 µl Plasmid (PM1 or PM2) Tap Tube Gently NO PLASMID ADDED ICE HEAT ICE 45 Second Heat Shock ICE HEAT ICE 45 Second Heat Shock 100 µl on LB/amp (+) Plate 100 µl on LB/amp (-) & LB (-) (-) Plates LB/amp (+) LB/amp (-) LB only (-)