Nt1310 Lab 1
KEY: ABE Lab 1:
Prelab:
Page A17, Questions 1 and 2:
1. What does the term genetic disease mean? What examples of generic diseases do you know about?
A genetic disease is a disease caused by an absent of defective gene in the DNA. Students may know of sickle cell anemia, which is a disease resulting from defective red blood cells; treatment of sickle cell anemia focuses primarily on relieving pain and controlling infections and other complications that may occur.
2. Adding human DNA to bacteria makes it possible to make human insulin. What do you already know about DNA? Be as detailed as possible and discuss the location of DNA in the cell, DNA structure, the replication of DNA, and the components of DNA.
Location of DNA in the cell: in eukaryotic cells, DNA is found in chromosomes in the nucleus and in mitochondria and chloroplasts. In prokaryotic cells, …show more content…
DNA is found in a chromosome in the nucleoid and in plasmids.
DNA structure: DNA is made up of two long molecules (strands) that are wrapped around each other in a double helix. Each DNA strand is made up of four nucleotides that are covalently bonded. The nucleotides are attached to each other be a sugar-phosphate backbone and have nitrogenous bases jutting out. The bases in one strand are hydrogen-bonded to bases in the other strand, and it is this bonding that creates the double helix. There are four bases and two possible base pairs. Bases that hydrogen-bond to each other are called complementary bases.
Replication of DNA: The two strands in the double helix are unwound and separated from each other. The nucleotides in each strand are hydrogen-bonded to complementary single nucleotides. The enzyme DNA polymerase then creates a new strand of DNA be ensuring that the single nucleotides are attached to each other by a sugar-phosphate backbone. The result is two double helices that are identical to the original double helix, except for rare copying errors.
Components of DNA: In addition to genes, DNA contains sequences that regulate and control the expression of genes, and sequences that control the structure of chromosomes.
Page A19, Question 1:
1. Why do you think it is necessary to use very small and exact volumes of reagents in biotechnology?
In this field you would use very small amounts of the reagents and the correct measurements of reagent amounts is necessary for procedures to be successful.
Page A25, Questions 1 and 2:
1. In what circumstances might it be important to use gel electrophoresis to separate and identify plasmids and short linear pieces of DNA?
This would be important if you are making a recombinant plasmid and have to verify that you have been successful.
2. Read through the Methods section on page A-26 through A-29 [of the Student Guide] and briefly outline the steps for Part A and for Part B, using words and a flowchart.
Laboratory 1.2, Part A Flowchart
Laboratory 1.2, Part B Flowchart
Page A30, Questions 1 and 2:
1. What is the importance of micropipettes and gel electrophoresis in the genetic engineering process?
Micropipettes are used to transfer very small and exact volumes of reagents and gel electrophoresis is used to separate and identify plasmids and short linear pieces of DNA
2. During the labs, you were often reminded to avoid contact with the pipette tips- for example, you were asked to put the pipette tip on without using your hands, to avoid setting gown the micropipette, to use the ejector button to remove the tip, and to keep the tip box closed. If you were working with plasmids and bacterial cells, why would these precautions be important?
When working with bacterial cells and plasmids, you would want to be very careful to avoid any cross-contaminations from occurring.
3. Study your gel electrophoresis results:
a) Which solution sample contained a single dye: S1, S2, or S3? How do you know? S3 contained a single dye because it only had one band.
b) The molecular weights for the dyes are 452.38 atomic units (au) for orange G, 669.98 au for bromophenol blue, and 538.62 au for xylene cyanole. How do these weights compare with your original conclusions about the weights of the dyes?
Base on the order of movement of the dyes through the gel (yellow moved the farthest, then purple, then blue), students should have originally predicted that the orange G (yellow dye) was the smallest, then bromophenol blue (purple), then xylene cyanole (blue) was the largest. But based on the molecular weights above, the purple was actually bigger than the blue, yet it moved farther in the gel. View the image below.
4) Do you think there was a difference in the amount of charge on one of the dye molecules? Explain the reasoning for your
response.
Yes, there must have been a difference in charge in the purple dye, or else it wouldn’t have moved farther than the smaller blue molecule. See the description below.
Prelab:
Page A17, Questions 1 and 2:
1. What does the term genetic disease mean? What examples of generic diseases do you know about?
A genetic disease is a disease caused by an absent of defective gene in the DNA. Students may know of sickle cell anemia, which is a disease resulting from defective red blood cells; treatment of sickle cell anemia focuses primarily on relieving pain and controlling infections and other complications that may occur.
2. Adding human DNA to bacteria makes it possible to make human insulin. What do you already know about DNA? Be as detailed as possible and discuss the location of DNA in the cell, DNA structure, the replication of DNA, and the components of DNA.
Location of DNA in the cell: in eukaryotic cells, DNA is found in chromosomes in the nucleus and in mitochondria and chloroplasts. In prokaryotic cells, …show more content…
DNA is found in a chromosome in the nucleoid and in plasmids.
DNA structure: DNA is made up of two long molecules (strands) that are wrapped around each other in a double helix. Each DNA strand is made up of four nucleotides that are covalently bonded. The nucleotides are attached to each other be a sugar-phosphate backbone and have nitrogenous bases jutting out. The bases in one strand are hydrogen-bonded to bases in the other strand, and it is this bonding that creates the double helix. There are four bases and two possible base pairs. Bases that hydrogen-bond to each other are called complementary bases.
Replication of DNA: The two strands in the double helix are unwound and separated from each other. The nucleotides in each strand are hydrogen-bonded to complementary single nucleotides. The enzyme DNA polymerase then creates a new strand of DNA be ensuring that the single nucleotides are attached to each other by a sugar-phosphate backbone. The result is two double helices that are identical to the original double helix, except for rare copying errors.
Components of DNA: In addition to genes, DNA contains sequences that regulate and control the expression of genes, and sequences that control the structure of chromosomes.
Page A19, Question 1:
1. Why do you think it is necessary to use very small and exact volumes of reagents in biotechnology?
In this field you would use very small amounts of the reagents and the correct measurements of reagent amounts is necessary for procedures to be successful.
Page A25, Questions 1 and 2:
1. In what circumstances might it be important to use gel electrophoresis to separate and identify plasmids and short linear pieces of DNA?
This would be important if you are making a recombinant plasmid and have to verify that you have been successful.
2. Read through the Methods section on page A-26 through A-29 [of the Student Guide] and briefly outline the steps for Part A and for Part B, using words and a flowchart.
Laboratory 1.2, Part A Flowchart
Laboratory 1.2, Part B Flowchart
Page A30, Questions 1 and 2:
1. What is the importance of micropipettes and gel electrophoresis in the genetic engineering process?
Micropipettes are used to transfer very small and exact volumes of reagents and gel electrophoresis is used to separate and identify plasmids and short linear pieces of DNA
2. During the labs, you were often reminded to avoid contact with the pipette tips- for example, you were asked to put the pipette tip on without using your hands, to avoid setting gown the micropipette, to use the ejector button to remove the tip, and to keep the tip box closed. If you were working with plasmids and bacterial cells, why would these precautions be important?
When working with bacterial cells and plasmids, you would want to be very careful to avoid any cross-contaminations from occurring.
3. Study your gel electrophoresis results:
a) Which solution sample contained a single dye: S1, S2, or S3? How do you know? S3 contained a single dye because it only had one band.
b) The molecular weights for the dyes are 452.38 atomic units (au) for orange G, 669.98 au for bromophenol blue, and 538.62 au for xylene cyanole. How do these weights compare with your original conclusions about the weights of the dyes?
Base on the order of movement of the dyes through the gel (yellow moved the farthest, then purple, then blue), students should have originally predicted that the orange G (yellow dye) was the smallest, then bromophenol blue (purple), then xylene cyanole (blue) was the largest. But based on the molecular weights above, the purple was actually bigger than the blue, yet it moved farther in the gel. View the image below.
4) Do you think there was a difference in the amount of charge on one of the dye molecules? Explain the reasoning for your
response.
Yes, there must have been a difference in charge in the purple dye, or else it wouldn’t have moved farther than the smaller blue molecule. See the description below.