Lucci_Prelab11_Monday2pm

Ampicillin broad spectrum semi synthetic penicillin

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Ampicillin – Broad-spectrum semi-synthetic penicillin that will kill a number of Gram- positive and Gram-negative bacteria. Plasmid – Small circular DNA molecules that exist apart from the chromosomes in most bacterial species. 22. Name the plasmid to be used in this exercise. pUC18 23. Study fig. 11.5. a) Which is the gene of interest for this exercise? Ampicillin-resistance b) What does this gene code for? It codes for the enzyme betalactamase (penicillinase) which inactivates ampicillin & other penicillins c) What is the size of this plasmid (in bp)? 2686bp 24. What is the bacterium to be used in this exercise? Escherichia coli 25. a) What does it mean to make a bacterium “competent”? The bacteria is able to accept/take-in the plasmid. b) What substance will be used to make the bacterium competent? CaCl 2 26. What volume of each of the following should you pipette? Total?? a) plasmid: 20 μ L (10 μ L x 2 tubes) b) competent bacterium cells: 200 μ L (100 μ L x 2 tubes) c) nutrient broth: 1.4 mL (700 μ L x 2 tubes) d) mixed bacterial suspension: 0.5 mL (250 μ L x 2 tubes)
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27. What is the purpose of each of the following? a) after adding plasmid, placing the tubes in 37 0 C for 5 minutes? The heat shock facilitates the uptake of plasmid DNA. b) after adding nutrient broth, placing the tubes in 37 0 C for 30 minutes? Allows the bacteria time to recover from the CaCl 2 treatment and to begin to express the ampicillin-resistance gene on the plasmid. 28. How many days later should you come back and check for growth on your plates? 1-2 days
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