Laboratory Procedure for bacterial transformation with pGLO

Laboratory Procedure forbacterial transformation with pGLO It’s glowing

Background • GFP (green fluorescent protein) comes from a jellyfish; Aequorea victoria • Causes bioluminescence (glowing) • Bacteria contain plasmids • Circular pieces of DNA that can be used to transfer genes from one organism to another

The purpose • Learn the principles of bacterial transformation • Transfer genes from a plasmid into the bacteria E.coli • Describe how to recognize a transformation has occurred • Explain the usefulness of this technique in other applications

Safety • E coli is a bacteria • Keep work areas clean • Practice sterile techniques • Wear gloves • Wash you hands before leaving lab

Assignments • You will be assigned to one of the following groups; • LB –plasmid (control) • LB +plasmid (control) • You are responsible for ALL procedures and ALL results from both groups

Procedures • Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your names or initials • Open the tubes, and using a sterile transfer pipet, transfer 250 µL of transformation solution (CaCl2) into each tube. Place on ice

Use a sterile loop to pick up a single colony of bacteria from your starter plate. • Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. • Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution. • Place the tube back on the ice. • Using a new sterile loop, repeat for the –pGLO tube. Close the –pGLO tubeand place on the ice

Use a sterile inoculating loop to add on loopful of plasmid DNA to the +plasmid tube • DO NOT add to the –plasmid tube • Return the tube to the ice • Both tubes must now incubate for 15 minutes

While the tubes are incubating, label your Petri dishes (on the bottom) as follows: • Group names • Date • 1.+plasmid LB/AMP (this is the exp. Group) • 2. -plasmid LB/AMP (this is the control group) • 3. Either: + plasmid LB OR –plasmid LB based on your assigned group

Heat shock • After 15 minutes of incubation • Remove BOTH tubes from the ice and immediately immerse in a 400 C water bath for 90 seconds • Gently swirl tubes while in the water bath • Remove after 90 seconds and return to ice for 1 minute

Use a sterile pipet to add 250 µL of Luria broth (LB) to each tube • Gently tap the tubes to mix the LB with the suspension • Place the test tubes in a rack for a 5-15 minute recovery

Cells from the –plasmid tube will be spread on the –plasmid plates • Cells from the +plasmid tubes will be spread on the +plasmid plates

Plating • Clamshell or slightly open the Petri dish • Using a sterile pipet add 100 µL from the correct tube and place onto the Petri dish • Pour 4-6 glass beads onto the plate surface • Use a back-forth motion (not round and round) to spread the suspension on the plate surface • To remove the glass beads, hold over the container and gently tap beads out

Wrap it up • Wrap the plates up with tape and write your initials on the tape • Place upside down in the incubator at 370 C for 24 hours. • This concludes Day 1 • Make sure to record any quantitative observations you made in this part of the lab

Day 2 • Remove your plates from the incubator • Examine the plates • Count each bacterial colony by marking it with a permanent marker • Record your results on your lab data sheet • Place the plates under the UV light to determine if they “glow” • Record the results

Clean-up • Clean up as instructed • Make sure to wash your hands before leaving lab

Lab Analysis • Complete the lab analysis questions for HW • Your teacher will discuss further lab requirements

Laboratory Procedure for bacterial transformation with pGLO