Intro
This lab invovles a process known as genetic transformation. Genetic transformation means a change caused by genes, and involves the insertion of a gene into an organism in order to change the organisms trait. In this lab we ware transforming the bacteria that codes for Green Fluorescent Protein (GFP). In nature, this protein can be found in the bioluminescent sea-jelly Aequorea victoria. Genetic transformation includes inserting new DNA into E.Coli cells. Bacteria often contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for more than trait. Scientists use a process called genetic engineering to insert coding for new traits into a plasmid. The new genetically engineered plasmid can be used in genetically transforming bacteria to give them the new trait. Procedure
1. Label 1 micro test tube +pGLO and another -PGLO.
2. Using a sterile transfer pipette, transfer 250 microliters of transformation solution (CACl2)
3. Place tubes on ice for 5 minutes.
4. Using a sterile loop, pick up one colony of bacteria from the starter plate. Place bacteria into +pGLO tube. Make sure all of the bacteria gets into tube.place tube in rack in ice. Do the same for -pGLO.
5. Add 10 microliters of plasmid DNA directly to liquid in +pGLO tube only. Flick tube to mix. Close tube and return to rack on ice. Do not add plasmid DNA to -pGLO tube.
6. Incubate tubes on ice for 10 minutes.
7. While tubes are on ice, label four Agar plates: LB/Amp plate +pGlO, LB/Amp/Ara +pGLO, LB/Amp -pGLO, LB -pGLO.
8. Heat Shock. Using foam rack, transfer both (+) (-) pGLO's into water bath set at 42 degrees C for exactly 50 seconds. After 50 seconds, place tubes back on ice. For best results, the change from heat to cold must be quick. Incubate tubes on ice for 2 minutes.
9. Remove tubes from ice and with a new sterile pipette add 250 microliters of LB nutrient broth in both +pGLO and -pGLO tubes.
10. Using sterile pipette, pipette 100 microliters of the transformation and control suspensions onto the appropriate plates.
11. Using a new sterile loop for each plate, spread the suspensions evenly around the surface of Agar.
12. Stack plates upside down and place them into the 37 degree C incubator until the next day.
8. Heat Shock. Using foam rack, transfer both (+) (-) pGLO's into water bath set at 42 degrees C for exactly 50 seconds. After 50 seconds, place tubes back on ice. For best results, the change from heat to cold must be quick. Incubate tubes on ice for 2 minutes.
9. Remove tubes from ice and with a new sterile pipette add 250 microliters of LB nutrient broth in both +pGLO and -pGLO tubes.
10. Using sterile pipette, pipette 100 microliters of the transformation and control suspensions onto the appropriate plates.
11. Using a new sterile loop for each plate, spread the suspensions evenly around the surface of Agar.
12. Stack plates upside down and place them into the 37 degree C incubator until the next day.
Results
Control
A-
ReplyDeleteVery good background info, but be sure to mention controls, variables ,and predictions