What is the purpose of putting ampicillin into the LB plates?
Ampicillin is an antibiotic and works by preventing E. coli from constructing cell walls, thereby killing the bacteria. When the ampicillin-resistance gene is present, it directs the production of an enzyme that blocks the action of the ampicillin, and the bacteria are able to survive.
Why do we add ampicillin in culture medium?
It is added to the culture for the best survival of culturing cells during our experiment. If your vector has ampR gene that codes for b-lactamase, then you’d add ampicillin to screen positives. Other reason is, amp is a broad range bacteriostatic antibiotic, which discourages contaminating bacteria from growing.
What is the primary purpose of using an ampicillin containing medium?
The purpose is to successfully transform the Escherichia coli bacteria when we expose it to extracellular plasmid DNA that contains the pGreen gene and the gene for ampicillin resistance.
How much ampicillin do I add to agar plates?
Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plate with a single red line on the side. Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml.
What is the purpose of LB amp ARA plate containing arabinose in addition to ampicillin?
The use of media containing ampicillin allows for the selection of successful transformants, however it is only when the sugar arabinose is also added to the media that it will be possible to observe the bright green fluorescence. This is because the GFP protein is under the control of an arabinose operon.
Why do you plate the cells from the viable count on LB agar without ampicillin?
If the bacteria are viable on the LB/amp plate, then they are resistant to ampicillin. If no bacterial colonies survive, then they were not ampicillin resistant (they were ampicillin sensitive). The UV light is necessary to cause the GFP protein within the bacteria to fluoresce.
What is the purpose of the antibiotics in the agar plate?
The addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic – usually conferred by a plasmid carrying the antibiotic resistance gene.