Laboratory Procedure for bacterial 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 tube and 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 40 0 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 37 0 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