Experiment 2- Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector “Table 1: Fragment Lengths” “DNA...

Experiment 2- Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector

“Table 1: Fragment Lengths”

“DNA Type”	“Longest Length (in base pairs)”
“Foreign”	720
“Plasmid”	2804

1. What is the expected size of the plasmid plus the cut foreign DNA?

2. What type of ends to the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?

3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?

4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?

Expert Solution

1.The expected size is- 3524 basepairs.

2. 1st part: BamHI and EcoR1 produce STAGGERED ENDS.

2nd part: in cloning experiment, when a researcher wants to insert a foreign DNA in a plasmid, he/she prefers the use of restriction enzymes that produce staggering ends/sticky ends. This is because when the insert DNA molecule and the foreign DNA molecule both have their 2 ends sticky, in presence of the DNA ligase enzyme, the two DNA molecule just sticks to one another- (refer to the figure). In contrast, when the ends are blunt, there arises issue because the insert DNA molecule can get inserted in either direction and can also attach with its own instead of with the target-creating plasmid that is non-functional.

3. The enzyme that is needed is -DNA ligase. The DNA ligase actually attaches the exposed 5'-Phosphate group present at the end of one DNA molecule with the exposed 3'-OH group of the other DNA molecule and catalyses the formation of a phosphodiester bond between them. This reaction requires the presence of ATP or NAD.

4. If there are no common restriction sites in the two DNA molecule(the plasmid and the insert DNA), then an adaptor molecule can be attached with the two ends of the insert DNA Molecule(flanking in the two ends)- that would have a desired restriction site. Once attached, the desired restriction enzyme can be used

gladiator answered 2 years ago

Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector” “Table 1: Fragment Lengths” DNA TYPE: LONGEST...

Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector” “Table 1: Fragment Lengths” DNA TYPE: LONGEST LENGTH FOREIGN: 716 PLASMID: 2837 Post-Lab Questions 1. What is the expected size of the plasmid plus the cut foreign DNA? 2. What type of ends to the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning? 3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does...

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector Foregin DNA = 720 Plasmid...

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector Foregin DNA = 720 Plasmid DNA 2804 Post-Lab Questions 1. What is the expected size of the plasmid plus the cut foreign DNA? 2524 base pairs 2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning? 3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does...

Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector Bacteria frequently contain extrachromosomal plasmids,...

Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector Bacteria frequently contain extrachromosomal plasmids, which are circular DNA genetic elements that are self-replicating. Plasmids are typically not necessary for the bacteria's survival but can sometimes confer a growth advantage to the bacteria. Plasmids can be transferred between bacteria and recombinant DNA technology makes use of this feature to introduce “foreign” genes into bacteria and use the bacteria as a cell “factory” to produce the gene. Genes are inserted...

Discuss the following research techniques 1. DNA ligase 2. Plasmid vector

If cloning of a 0.8 kb DNA segment was performed at the PstI site of plasmid...

If cloning of a 0.8 kb DNA segment was performed at the PstI site of plasmid pBR322 and the E. coli strain HB-101 was transformed with the construct: a) What is the selection criteria for E. Coli transformants with the recombinant plasmid ?: -Ecoli growth in nutrient-rich culture medium____ -Ecoli growth in culture medium rich in nutrients plus ampicillim_____ - Growth of E. coli in a culture medium rich in nutrients plus tetracycline_____ -Ecoli growth in culture medium rich in...

1.) Describe the four stages of genetic engineering: cleave DNA & insert into cloning vector -producing...

1.) Describe the four stages of genetic engineering: cleave DNA & insert into cloning vector -producing recombinant DNA, insert recombinant vector into bacterial cells -cloning, amplification of recombinant vector, screening for recombinant DNA. 2.) What and how are antibiotic resistance and the lacZx gene used in screening? 3.) What is Agrobacterium tumefaciens? What normally occurs upon Agrobacterium tumafaciencs infection in plants? How is it used to genetically engineer plants (stably insert foreign genes into plants)?

Genetics Essay 1. Describe DNA vector cloning. In your description, make sure to include: a) the...

Genetics Essay 1. Describe DNA vector cloning. In your description, make sure to include: a) the process (steps) b) three things that a plasmid cloning vector must have c) an explanation of how blue and white screening works during DNA recombinant technology

1.How do reproductive cloning and therapeutic cloning differ? a. The DNA in the nucleus of cells...

1.How do reproductive cloning and therapeutic cloning differ? a. The DNA in the nucleus of cells produced for therapeutic cloning is genetically identical to the donor genome, whereas in cells produced for reproductive cloning it is not. b. Reproductive cloning requires a supply of fertilized donor egg cells, whereas therapeutic cloning requires unfertilized egg cells. c. Therapeutic cloning requires nuclear transplantation, whereas reproductive cloning does not. d. Embryos are placed into foster mothers during reproductive cloning but not during therapeutic...

If you would do a PCR experiment using the Klenow fragment of the E.coli DNA polymerase...

If you would do a PCR experiment using the Klenow fragment of the E.coli DNA polymerase I instead of Taq DNA polymerase, what would you have to change in the experimental set-up and explain why?

Experiment 3: DNA Extraction 1. What is the texture and consistency of the DNA? 2. Why...

Experiment 3: DNA Extraction 1. What is the texture and consistency of the DNA? 2. Why did we use a salt in the extraction solution? 3. Is the DNA soluble in the aqueous solution or alcohol? 4. What else might be in the ethanol/aqueous interface? How could you eliminate this? 5. Which DNA bases pair with each other? How many hydrogen bonds are shared by each pair? 6. How is information to make proteins passed on through generations? 7. Watch...

Question