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MOL BIO LAB ASSIGNMENT # 1
Due Feb. 26th, 2009
A. GROWING BACTERIA AND YEAST
1. Streak Plate Experiment
Examine your streak plates for growth and for the presence of discrete, isolated colonies.
a. Describe the physical characteristics of the two types of colonies from the
E. coli and Saccharomyces cerevisiae cultures.
The E. coli culture (MC4100), plated on LB plates, and Saccharomyces cerevisiae
culture (FY2031 - yeast), plated on YEPD plates, both contained numerous round,
milky-colored colonies.
b. What organism prefers LB plates? What organism prefers YEPD plates?
E. coli prefers nutrients in the LB agar plates, while Saccharomyces cerevisiae prefers
those in the YEPD plates.
2. Culture transfer: Examine the inoculated broth for growth.
a. How can you tell whether or not bacteria have grown in the LB broth?
If bacteria have grown in the inoculated LB, there will be noticeable turbidity
(cloudiness) throughout the broth. The absence of bacterial growth will result in clear
broth (as it was before inoculation).
b. Was growth present in the broth to broth transfer? Explain.
B. ISOLATING PLASMID DNA- RAPID BOILING METHOD AND ALKALINE LYSIS
METHOD
Please type the answers to questions 2 and 4 on a separate sheet of paper. Your flow charts from
questions 1 and 3 can be hand-written as can your calculations for questions 5.
1. Prepare a simple flow chart showing the steps in the rapid boiling method from Exercise 4,
p.31. Start with the strain we used: XL1 pRY121.
SEE ATTACHED FLOWCHART
2. What was the purpose of the following reagents in the rapid boiling method: lysozyme and
isopropanol?
Lysozyme is added to the solution containing the E. coli cells and lysis buffer (weakens outer
membrane of bacterium) and is used to destroy the bacterium’s small peptidoglycan layer and
open up the bacterial cells (cell membrane lysis). Isopropanol is used in conjunction with salt
(sodium acetate) to precipitate the plasmid DNA from the supernatant.
3. Prepare a simple flow chart showing the steps in the alkaline lysis prep done with the Qiagen
Plasmid Miniprep Kit. Start with the strain we used: XL1 pRY121.
SEE ATTACHED FLOWCHART
4. We were interested in isolating only the plasmid DNA.
a. What is the name of the plasmid?
The name of the plasmid from E. coli cells is pRY 121.
b. How did we get rid of the chromosomal DNA in the rapid boiling method? In the
alkaline lysis method?
In the rapid boiling method, the addition of lysis buffer to cell pellet weakens the cellular
membrane for easy access for lysozymes to the peptidoglycan layer. Then lysozyme solution is
added, which destroys the peptidoglycan layer and breaks open the bacterial cells to release its
contents (mainly chromosomal and plasmid DNA). Centrifuging then separates the
chromosomal DNA along with other cellular debris (cellular membrane) into a pellet, while the
plasmid DNA is present in the resulting supernatant.
5. In lab, we often need TE buffer, which is10mM Tris and 1mM EDTA. You are given two
stock solutions of 0.1M Tris and 0.2M EDTA and are asked to make up 100ml of TE buffer.
Tell how many milliliters of the Tris and the EDTA stocks and distilled water that you will
use in preparing this buffer. Remember C1 V1 = C2 V2.