What conclusions can you make when comparing the LB pGLO and LB AMP pGLO plates?
The (-) pGLO/LB control plate can be compared to any of the LB/amp plates to show that plasmid uptake is required for the growth in the presence of ampicillin. The (-) pGLO LB/amp plate shows that the starter culture does not grow on the LB/amp plate.
What is the purpose of pGLO transformation lab?
pGLO Bacterial Transformation and GFP Kits pGLO Lab Kits utilize Bio-Rad’s pGLO plasmid, which encodes a green fluorescent protein (GFP), to enable instructors to give students a hands-on introduction to transformation, cloning, protein chromatography, and electrophoresis techniques.
What is the independent variable in the pGLO lab?
Independent variable: the addition of pGLO, arabinose, and ampicilin. Dependent variable: bacterial growth and if it glows.
Why would we expect to see no colonies on the pGLO LB amp plate?
This is because the +pGLO was given the plasmid which gives it resistance to the antibotic in its plate, while the -pGLO was not. So therefore you can prove that a genetic transformation occurred because the bacteria in the +pGLO LB/amp plate would survive. 4.
What three genes have been removed in the pGLO plasmid?
The pGLO plasmid contains both the promoter (pBAD) and araC gene, but araB, araA, and araD have been replaced by the single gene that codes for GFP, which serves as a reporter gene.
Why does pGLO needed arabinose?
When arabinose is added to E. coli cells that contain pGLO, the GFP gene will be highly expressed, becoming one of the most abundant proteins in the cell. When arabinose is not present, the amount of GFP expression will be close to zero.
What purpose does spreading pGLO on the LB plate serve what might cause your cells to fail to grow on the LB plate?
6) What purpose does spreading –pGLO on the LB plate serve? What might cause your cells to fail to grow on the LB plate? The purpose of spreading pGLO on the LB plate was to enable bacteria to grow, otherwise the ampicillin would kill them. If the pGLO is not spread, then the cells would be killed by the ampicillin.
How does the pGLO plasmid work?
The pGLO plasmid was engineered to allow inducible expression of GFP. “Inducible” means that the cells will only produce GFP when an inducer (in this case, the sugar arabinose) is added. This level of control was achieved by connecting the GFP coding region with a bacterial promoter.
What is the importance of the ampicillin resistance gene in the pGLO plasmid?
The pGLO plasmid also contains a gene for ampicillin resistance so that successful transformants can be distinguished from cells that have not taken up the plasmid DNA by their ability to grow on a medium containing this antibiotic.
What is the purpose of the ampicillin resistance gene on the plasmid in the pGLO experiment?
Other pGLO features In addition to the GFP operon, you should understand two other elements of the pGLO plasmid: bla — also known as the ampicillin resistance gene; a gene that encodes the enzyme β-lactamase, which breaks down the antibiotic ampicillin.
What is a good transformation efficiency value?
Transformation efficiency and cloning applications For most cloning applications, a transformation efficiency between 106 and 1010 CFU/µg is considered adequate. Lower transformation efficiencies of approximately 106 CFU/µg can work well for routine cloning and subcloning experiments with supercoiled plasmids.
How do you know if a cell is competent?
therefore you can use plasmid DNA which you can buy and then checking with a definite concentration (10 pg DNA) and then you can see after transformation the competent. If you have competent cells in your lab look into the kit, often there is a control plasmid DNA inside.