Homework Answers
7) SaII site is present in the tetracycline resistance gene for cloning plasmid is digested with SaII and there the insert has to be cloned this disrupts the tetracycline resistance gene so after cloning tetracycline resistance is lost so to select cloned vectors only ampicillin has to be used.
8) let`s do the digestion with BamHI then is the insert is
present bands produced are of 600bp, 800bp, and 4661 bp
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7. Explain the procedure for cloning DNA fragment into the plasmid PBR322 (shown on the right) (S...
A student clones a DNA fragment into the Sall site of pBR322. Sall is a restriction...
A student clones a DNA fragment into the Sall site of pBR322. Sall is a restriction enzyme and pBR322 is the name of a plasmid (diagram below). Predict the media on which she could expect growth of E. coli containing the recombinant plasmid. Explain your answers. (16 pts) 2. EcoRI BamHI Pst Sall Ampicillin Tetracycline resistance (AmpR) resistance (TetR) pBR322 (4,361 bp) Origin of replication (ori) Pvull Media nutrient agar with ampicilin nutrient agar with tetracycline nutrient agar with ampicillin...
Q5. Figure 2 shows the plasmid PGL4.83[hRlucP/Puro]. Based on Figure 2, answer the following: Synthetic poly(A)...
Q5. Figure 2 shows the plasmid PGL4.83[hRlucP/Puro]. Based on Figure 2, answer the following: Synthetic poly(A) Amp' Sall 2599 ori Sfil Acc651 Kpnl EcolCRI Sacl Nhel Xhol Bglli D Synthetic poly(A) PGL4.83[hRlucP/Puro) Vector (4815bp) Sfil Hindlll hRlucP Puro SV40 early enhancer/ promoter EcoRI 1033 SV40 late poly(A) signal 5141MA 1431 BamHI Figure 2 (Source: httpwww.biofeng.comzaitīburup GL4.83.html 1) There are two synthetic polv(A) sites flanking the ori and Amp genes. State the technique used to achieve these poly(A) sites. Briefly elaborate...
Chromosomal and plasmid DNA can be cut into manageable pieces by restriction enzymes. Using agarose gel...
Chromosomal and plasmid DNA can be cut into manageable pieces by
restriction enzymes. Using agarose gel electrophoresis, the DNA
fragments can be separated on a gel, based on their lengths. In
order to see the fragments, a stain is typically added to the gel.
The size of each fragment can be determined by comparing each one
to a DNA molecular weight marker of known size.
Below is a map of pBR22 plasmid. The position and base pair
number of the...
8. DNA cloning. The first DNA clone using recombinant DNA technology was human insulin, which was...
8. DNA cloning. The first DNA clone using recombinant DNA technology was human insulin, which was made in 1978. The insulin gene was inserted into a plasmid and the gene was transcribed and translated from the plasmid in E coll cells. Insulin is synthesized by ribosomes in human pancreatic cells. Insulin is synthesized as a longer polypeptide, which is then trimmed by proteases to make the mature chains A and B (see Fig. 2.17 on page 39 of your textbook)....
III. Subclone the gene into plasmid, extract the plasmid DNA. 5. You know that your insert...
III. Subclone the gene into plasmid, extract the plasmid DNA. 5. You know that your insert (gene of interest, GOI) is flanked by the EcoRI sites, which makes this restriction enzyme a perfect candidate to cut out your gene. You also know that the GOI has a unique BamH1 restriction site. After subcloning the PCR product into the plasmid, a purified DNA preparation of the plasmid is digested to completion with BamHI restriction endonuclease. In separate reactions, the same preparation...
1. Molecular cloning has made it possible to take a DNA fragment from nuclear DNA, containing...
1. Molecular cloning has made it possible to take a DNA fragment from nuclear DNA, containing an entire gene, and insert it into a cloning plasmid just behind a phage promoter. This means that the cloned gene can be transcribed in a test tube using a commercially available phage RNA polymerase protein. If you were to use agarose gel electrophoresis to separate RNA according to size, would the RNA produced in the test tube show any differences with RNA from...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification...
The features of two plasmids, pkan-GFP and PBS, are illustrated below. poperon MCS LacZ enc (a-fragment)...
The features of two plasmids, pkan-GFP and PBS, are illustrated below. poperon MCS LacZ enc (a-fragment) pKan-GFP 10,000 bp PBS 5,500 bp Ori pkan-GFP has a 6 kbp promoterless gfp operon that encodes a green-fluorescent protein, which glows green. Kan" is a gene (500 bp) that provides resistance to the antibiotic kanamycin. PBS contains the lacZ' gene (which has its own promoter) and a 100 bp multiple cloning site (MCS) located within the lacZ'gene. Tet" is a gene (1,500 bp)...
A1. The following is the DNA sequence of a hypothetical gene for the SMALL protein. It...
A1. The following is the DNA sequence of a hypothetical gene for the SMALL protein. It is called the SMALL gene. i atgggattac actgtcacga ccaaatagcc ttcattgtat 41 caaaaggato aatcgagtta tag Imagine you are doing a research project in a laboratory and your supervisor asks you to clone the SMALL gene into the PBR322 plasmid (shown below). You must use the Pstl and EcoRI sites for your cloning. HindIII EcoRI | EcoRV BamHI 4359 0 29 185 4000 375 Sall Psti...
You are preparing to clone a DNA fragment into a plasmid vector. You start by linearizing...
You are preparing to clone a DNA fragment into a plasmid vector. You start by linearizing your plasmid (concentration = 500 ng/μl) with EcoRI, which is provided in a standard 50% glycerol solution at 10 units/μl. Your enzyme also comes with an appropriate 10X reaction buffer. Taking into account the final allowable glycerol concentration, you want to add the maximum amount of EcoRI, to achieve complete digestion of 1 μg plasmid DNA in a total volume of 20 μl. Fill...
A student clones a DNA fragment into the Sall site of pBR322. Sall is a restriction enzyme and pBR322 is the name of a plasmid (diagram below). Predict the media on which she could expect growth of E. coli containing the recombinant plasmid. Explain your answers. (16 pts) 2. EcoRI BamHI Pst Sall Ampicillin Tetracycline resistance (AmpR) resistance (TetR) pBR322 (4,361 bp) Origin of replication (ori) Pvull Media nutrient agar with ampicilin nutrient agar with tetracycline nutrient agar with ampicillin...
Q5. Figure 2 shows the plasmid PGL4.83[hRlucP/Puro]. Based on Figure 2, answer the following: Synthetic poly(A) Amp' Sall 2599 ori Sfil Acc651 Kpnl EcolCRI Sacl Nhel Xhol Bglli D Synthetic poly(A) PGL4.83[hRlucP/Puro) Vector (4815bp) Sfil Hindlll hRlucP Puro SV40 early enhancer/ promoter EcoRI 1033 SV40 late poly(A) signal 5141MA 1431 BamHI Figure 2 (Source: httpwww.biofeng.comzaitīburup GL4.83.html 1) There are two synthetic polv(A) sites flanking the ori and Amp genes. State the technique used to achieve these poly(A) sites. Briefly elaborate...
Chromosomal and plasmid DNA can be cut into manageable pieces by
restriction enzymes. Using agarose gel electrophoresis, the DNA
fragments can be separated on a gel, based on their lengths. In
order to see the fragments, a stain is typically added to the gel.
The size of each fragment can be determined by comparing each one
to a DNA molecular weight marker of known size.
Below is a map of pBR22 plasmid. The position and base pair
number of the...
8. DNA cloning. The first DNA clone using recombinant DNA technology was human insulin, which was made in 1978. The insulin gene was inserted into a plasmid and the gene was transcribed and translated from the plasmid in E coll cells. Insulin is synthesized by ribosomes in human pancreatic cells. Insulin is synthesized as a longer polypeptide, which is then trimmed by proteases to make the mature chains A and B (see Fig. 2.17 on page 39 of your textbook)....
III. Subclone the gene into plasmid, extract the plasmid DNA. 5. You know that your insert (gene of interest, GOI) is flanked by the EcoRI sites, which makes this restriction enzyme a perfect candidate to cut out your gene. You also know that the GOI has a unique BamH1 restriction site. After subcloning the PCR product into the plasmid, a purified DNA preparation of the plasmid is digested to completion with BamHI restriction endonuclease. In separate reactions, the same preparation...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification...
The features of two plasmids, pkan-GFP and PBS, are illustrated below. poperon MCS LacZ enc (a-fragment) pKan-GFP 10,000 bp PBS 5,500 bp Ori pkan-GFP has a 6 kbp promoterless gfp operon that encodes a green-fluorescent protein, which glows green. Kan" is a gene (500 bp) that provides resistance to the antibiotic kanamycin. PBS contains the lacZ' gene (which has its own promoter) and a 100 bp multiple cloning site (MCS) located within the lacZ'gene. Tet" is a gene (1,500 bp)...
A1. The following is the DNA sequence of a hypothetical gene for the SMALL protein. It is called the SMALL gene. i atgggattac actgtcacga ccaaatagcc ttcattgtat 41 caaaaggato aatcgagtta tag Imagine you are doing a research project in a laboratory and your supervisor asks you to clone the SMALL gene into the PBR322 plasmid (shown below). You must use the Pstl and EcoRI sites for your cloning. HindIII EcoRI | EcoRV BamHI 4359 0 29 185 4000 375 Sall Psti...
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