Prescott's Microbiology
10th Edition
ISBN: 9781259281594
Author: Joanne Willey, Linda Sherwood Adjunt Professor Lecturer, Christopher J. Woolverton Professor
Publisher: McGraw-Hill Education
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Chapter 36.4, Problem 4MI
Summary Introduction
To determine: The microorganism for which the patient who have donated the serum for the given (fig 36.13) Western blot has been exposed.
Introduction: Molecular biology technique which is used for detection of a particular sequence of DNA in samples is called Western blotting. This technique includes the transfer of DNA fragments which are separated by electrophoresis to a filter membrane followed by DNA fragment detection with the help of probe hybridization.
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1)Which plate did you see purple/pink/blue bacterial cells? Why did you see this growth? Explain your answer in terms of transformation and plasmids?
2) Calculating Transformation Efficiency
For the +DNA/+Amp/+IPTG plate, record the following:
Number of transformants (colonies): _________________
Nanograms of plasmid DNA added: 50 ng
Final recovery volume: 0.50 mL
Volume plated: 0.25 mL
Transformation efficiency equation:
Transformation efficiency = Number of transformants / µg of DNA x Final volume at recovery (mL)/ volume plated (mL)
3) Using the equation above, calculate the transformation efficiency.
4) Describe the success of the transformation efficiency of this demo based on the calculation you did above?
1) Look at the ideal results. Were your predictions accurate, and how did they compare with your results?
2) You used aseptic technique during this lab. Why is it important to work in a sterile manner when working with bacteria in the lab?
3) Why are the cells incubated at 42°C?
Overview of Transformation Protocol
-Prepare competent bacteria for transformation:
Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure.
Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed.
-Transformation procedure:
Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA.
Add CaCl2 to both tubes.
Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube.
Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes.
Add recovery broth to both tubes.
Incubate both tubes in a 37 C water bath for 5 minutes.
Questions:
1) What differences would you expect to see between the…
Chapter 36 Solutions
Prescott's Microbiology
Ch. 36.3 - You have detected aerobic, Gram-positive cocci...Ch. 36.3 - Prob. 2MICh. 36.3 - Prob. 1RIACh. 36.3 - Describe in general how biochemical tests are used...Ch. 36.3 - Prob. 3RIACh. 36.3 - Prob. 4RIACh. 36.3 - Describe a dichotomous key that could be used to...Ch. 36.3 - How can nucleic acidbased detection methods be...Ch. 36.3 - How can a suspect bacterium be fingerprinted?Ch. 36.4 - Prob. 1MI
Ch. 36.4 - Figure 36.10 Agglutination Tests. (a) Tube...Ch. 36.4 - Prob. 3MICh. 36.4 - Prob. 4MICh. 36.4 - Prob. 1RIACh. 36.4 - Prob. 2RIACh. 36.4 - Prob. 3RIACh. 36.4 - Prob. 4RIACh. 36.4 - Prob. 5RIACh. 36.4 - Name two types of immunodiffusion tests and...Ch. 36.4 - Prob. 7RIACh. 36.4 - Prob. 8RIACh. 36.4 - Prob. 9RIACh. 36 - As more ways of identifying the characteristics of...Ch. 36 - Prob. 2CHICh. 36 - ELISA tests usually use a primary and secondary...Ch. 36 - Legionella pneumophila is a bacterium that is...Ch. 36 - Prob. 5CHI
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