Insert genes from 1 organism into another
View genetic transformation in bacteria by glow and growth of colonies
Assess how efficient the transformation was
Everything will be in the lab notebook. DO NOT WRITE ON A SEPARATE SHEET OF PAPER.
We can have up to 8 groups (yes, if you want you can work by yourself).
There are pre-lab questions which I will check on Wednesday.
This lab takes a few days due to wait time for the plates to culture.
Things might not work the way they're supposed to but that's OK. It's science; things don't always have to work out!
Label 2 tubes: +pGLO and -pGLO
Add 250uL of transformation solution into each tube. Place tubes on ice.
Use a sterile loop to pick 2-4 large colonies of bacteria from the starter plate. Colonies to choose should be 1-2mm. Put the loop (with colonies) in the +pGLO tube and spin the loop so that the colonies mix into the solution. Place tube on ice.
Repeat step 3 with a new loop for -pGLO.
Put a new loop in the pGLO plasmid DNA stock tube. Obtain a loopful. Put this loop into the +pGLO tube ONLY! Put back on ice.
Make sure tubes are fully in ice and wait 10 mins for them to inoculate. During the wait, label the agar plates:
LB/amp plate ----> write "+pGLO"
LB/amp/ara plate ----> write "+pGLO"
LB plate ----> write "-pGLO"
LB/amp plate ----> write "-pGLO"
After 10 min, use a foam rack and transfer tube to a water bath for exactly 50 sec. Remove and QUICKLY return to the ice for 2 min.
After 2 min, take the tubes and add 250uL if nutrient broth to each tube. Incubate at room temperature for 10 min.
After 10 min, "flick" both tubes to resuspend the bacteria. With a pipette add 100uL of the +pGLO solution (transformation solution) to the plates labeled "+pGLO."
With a new pipette add 100uL of -pGLO solution (control solution) to the plates labeled "-pGLO."
Use a loop for the +pGLO plates and spread the solution evenly. See the instructions for an image of how to do this.
Repeat step 11 for the -pGLO plates with a new loop.
Stack all 4 plates and tape them together. Write you name on the bottom plate in the stack.
Flip the stack upside and place in the incubator at 37C (or 2-3 days at RT).
Observe growth under UV light. Draw what you see and write observations. Your observations need to include:
amount of bacterial growth
color under normal AND UV light
how many colonies on each plate (count how many you see)
Answer analysis questions
Calculate transformation efficiency and post questions.