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 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 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