Name

BIO 230 EXAM 3

Directions: Please answer the following questions using complete sentences, proper grammar, and preferably typed and double spaced. You may use your lab notebooks and the Edvotek 303 Protocol to help you with your procedures and answers. Point values are listed. Due Wed 12/20 @ 1:30 (and anytime BEFORE that is fantastic!) Lateness will cost you 10 points per day! 1. You have been lucky enough to be hired by Nuclea Biotechnologies and your boss has asked you to cast an 8% agarose gel and set up an electrophoresis machine to run a sample of DNA for separation. Please write the procedure necessary to complete the task your boss has asked you to do. Assume you are using a previously prepared TBE buffer. (10)

2. Explain in your own words the purpose of both the electrical current and agarose gel in electrophoresis. (5) 3. What characteristics of DNA make it a good candidate to be studies using gel electrophoresis? (5) 4. What is the natural host of the Green Fluorescent Protein (GFP)? (5) 5. What is the cyclic tripeptide responsible for the light emission of GFP? (5) 6. What is the one of the important visual application tools of the GFP? (5) 7. What does it mean to transform a bacterial cell? (10) 8. What does competency mean in reference to bacterial cells? (5) 9. What step(s) did we take to make our E. coli competent? (15) 10.Although we did not calculate transformation efficiency, what is the average transformation efficiency? (5) 11.What is a plasmid? (10) 12.Explain the benefit of bacterial plasmids containing the ampicillin resistance gene to the budding biotechnologist? (10) 13.What is SDA-PAGE and why did we use it in the Green Fluorescent Protein Lab? (10) This link will be great at helping with question. http://www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html

1. Assuming the gel bed is 7cm x 14cm, I would need 50mL of gel. An 8% agarose gel would have 4g of agarose in a 50mL volume. To set up the bed, I would close off the open ends of a

clean and dry gel bed (casting tray) by using rubber dams and then place a well-former template (comb) in the set of notches at the middle of the bed, making sure the comb sits firmly and evenly across the bed. Next, I would weigh out 4g of agarose. I would then add about 40mL of diluted (1x) gel buffer to a 100mL flask, add the 4g of agarose, and bring to volume while swirling to disperse any clumps. Before heating, I would cover the flask with aluminum foil to minimize evaporation. Then, I would heat the mixture to boiling over a hot plate with occasional swirling. I would continue to boil until all the agarose is completely dissolved and the final solution appears clear without any undissolved particles. Next, I would cool the agarose solution to 60C with careful swirling to promote even dissipation of heat. Using a transfer pipet, I would deposit a small amount of the cooled agarose to both inside ends of the bed and wait for about 1 minute for the agarose to solidify. Then, making sure the bed was on a level surface Id pour the cooled 60 C agarose solution into the bed and allow the gel to completely solidify. After about 20 minutes, when the gel was completely solidified, I would carefully and slowly remove the rubber dams from the gel bed and remove the comb by slowly pulling straight up to prevent tearing the sample wells. Then Id place the gel, on its bed, into the electrophoresis chamber, properly oriented, centered and level on the platform. After making sure the drainage holes in the chamber are plugged, I would fill the electrophoresis apparatus chamber with the diluted (1x) electrophoresis buffer making sure that the gel is completely submerged under buffer. Last, I would get the safety cover, leads and power source ready for after the samples have been loaded.

2. The electrical current pulls charged molecules in a sample through the gel. Negatively charged particles go towards one end and positively charged molecules go towards the other. The agarose gel has microscopic pores that affect the rate at which the molecules move towards one end or the other. Smaller molecules can move more easily through the pores, while larger molecules get stuck. The distance the molecules travel from the sample wells

toward one end or the other helps us determine the size or shape of the molecules and their relative charge.

3. Double stranded DNA moves through gel like a long rod. The uniformity of shape of DNA allows different DNA to be separated by its length in number of base pairs. The fewer the number or base pairs the farther the DNA will travel through the gel.

4. The jelly fish Aquorea victoria is the natural host of the green fluorescent protein.

5. The cyclic tripeptide responsible for the light emission of GFP is -Ser-65-Tyr-66-Gly-67-.

6. One of the important visual application tools of the GFP is the reporter gene construct. Reporter gene plasmids are created by ligating the GFP gene to a gene of interest and then introducing the reporter plasmid into a certain cell type, such as stem cells, where the GFP reporter gene construct is used to track their differentiation into different cell types during development of embryos.

7. Transforming a bacterial cell is inserting foreign plasmid DNA into bacterial cells where the plasmids replicate and express their genes.

8. Competency means that the bacterial cells have been chemically treated to be able to take in foreign DNA from their surrounding environment through the cell wall.

9. The E. coli was made competent by adding ice cold CaCl 2 to the samples and then heat shocking them at 42 C for 90 seconds.

10. The average transformation efficiency is 1600 transformants per g.

11. A plasmid is a small, circular, self-replicating double-stranded DNA molecule found primarily in bacterial cells.

12. Bacterial plasmids containing the ampicillin resistance gene can be used to transform bacteria. In antibiotic selection, the bacteria is grown in a liquid medium and then plated onto an agar medium containing ampicillin. Only the cells were transformed with the plasmids containing the ampicillin resistant genes will grow to produce colonies. 13. SDS-PAGE is sodium dodecyl sulfate polyacrylamide gel electrophoresis. SDS is a detergent that dissolves cell membranes releasing proteins into solution. SDS was used so that we could detect if the GFP was being expressed. The GFP had to be free in solution. Polyacrylamide gel was used because proteins are too big to be affected by the sieve effect in agarose gel. The polyacrylamide gel is better suited to see the differences in charge of proteins, which would determine how far they traveled in the gel.