Homework Answers
One of the desired location to insert the fragment in the plasmid is the multiple cloning site. Thus correct restriction enzymes must be chosen. pSAP has following components;
ori = region where DNA replication starts
multiple cloning sites for insertion of DNA fragment has restriction sites as given : EcoRI, BamHI,XhoI,KpnI, Nde1, Hind III
amp = a gene that allows cells to grow when exposed to certain conditions
Hence best restriction endonucleases include would be EcoRI and Hind III
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Suppose you have a dna fragement you would like to insert into the psap plasmid. the...
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...
After a plasmid vector has been linearized using one restriction endonuclease, DNA Ligase cannot recircularize the...
After a plasmid vector has been linearized using one restriction endonuclease, DNA Ligase cannot recircularize the plasmid unless it contains an insert DNA fragment to be cloned True or False True False
A genetics problem covering chapter 10 concepts. Could really use some help! The Notch gene, involved...
A genetics problem covering chapter 10 concepts. Could
really use some help!
The Notch gene, involved in Drosophila development, is contained within a restriction fragment of Drosophila genomic DNA produced by cleavage with the enzyme SalI. The restriction map of this Drosophila fragment for several enzymes (Sall, PstI, and Xhol) is shown here; numbers indicate the distances between adjacent restriction sites. This fragment is cloned by sticky-end ligation into the single Sall site of a bacterial plasmid vector that is...
Suppose you digest the genomic DNA of a particular organism with the restriction enzyme SauA. Then...
Suppose you digest the genomic DNA of a particular organism with
the restriction enzyme SauA. Then you ligate the resulting fragment
into a unique BamHI cloning site of a plasmid. The sequence of the
restriction sites and position of cleavage is shown below
Note: X and Y and their complementary bases Z and Y’
respectively can be any base (A,C, G, or T)
1) As you can see, ligation is possible because the two restriction
enzymes produce compatible sticky ends....
please help me with these questions Lab 8 Extension Activity: Plasmid Mapping and Restriction Enzymes Mapping...
please help me with these questions
Lab 8 Extension Activity: Plasmid Mapping and Restriction Enzymes Mapping the Plasmid The first step in mapping a plasmid is to determine how many times a restriction site is found on that plasmid. Examine the results for plasmid 55 as an example. The data given in the following table are for the double digest using EcoRI and Pstl. Also, giving are the data for single digests by the individual enzymes. The numbers in the...
In your previous prac session you digested your POTC-A plasmid DNA with 3 enzyme mixes (AB...
In your previous prac session you digested your POTC-A plasmid DNA with 3 enzyme mixes (AB and C). One mix contained the restriction endonuclease Kpnl alone, another contained both del and Not and another contained Sphi and Nhe, but you don't know which was which yet. The sites where these enzymes cut POTC-A are indicated on the plasmid map at the top of the page. Calculate the sizes of the DNA fragments that you would expect to see on your...
Suppose you are going to do a restriction digest with a plasmid, using the restriction enzyme...
Suppose you are going to do a restriction digest with a plasmid, using the restriction enzyme Eco R1. A map of the plasmid is shown here. The entire plasmid is 6000 bp, and there are Eco R1 restriction sites at 1500 bp, 2000 bp, and 4000 bp. You’re going to run the entire volume of the digest on a gel, and you want to cut just enough DNA to have 50 ng in the smallest band on your gel. Starting...
7. Explain the procedure for cloning DNA fragment into the plasmid PBR322 (shown on the right) (S...
7. Explain the procedure for cloning DNA fragment into the plasmid PBR322 (shown on the right) (S pts.). The gene fragment of interest was obtained by digestion of chromosomal DNA with the restriction enzyme Sall and subsequent purification using agarose gel electrophoresis. Which antiblotic would you use in the final step of the cloning procedure, and Pst why? EcoR Sal Ampicillin Tetracycline resistanica(Ter Amp) PBR322 4,361 bp) Origin of replicatiorn (ori Pvull 8. Assume that your gene fragment from question...
1. You perform a restriction endonuclease assay on an uncharacterized DNA molecule, using Pstl, Sall, and...
1. You perform a restriction endonuclease assay on an uncharacterized DNA molecule, using Pstl, Sall, and Xhol. When you run the DNA on a 1% agarose gel, you obtain the following information regarding fragment number and length (all lengths are in bp): Pstl (P) 700 200 Sall(s) 600 300 Xhol (X) 500 350 50 Pstl + Sall (P+S) 600 200 100 Pstl + Xhol (P+X) 500 200 150 50 Sall + Xhol (S+X) 500 250 100 50 Solve the following...
If you wanted to insert a gene into a plasmid, why would it be helpful to...
If you wanted to insert a gene into a plasmid, why would it be helpful to know the plasmid’s restriction enzymes cleavage sites?
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...
After a plasmid vector has been linearized using one restriction endonuclease, DNA Ligase cannot recircularize the plasmid unless it contains an insert DNA fragment to be cloned True or False True False
A genetics problem covering chapter 10 concepts. Could
really use some help!
The Notch gene, involved in Drosophila development, is contained within a restriction fragment of Drosophila genomic DNA produced by cleavage with the enzyme SalI. The restriction map of this Drosophila fragment for several enzymes (Sall, PstI, and Xhol) is shown here; numbers indicate the distances between adjacent restriction sites. This fragment is cloned by sticky-end ligation into the single Sall site of a bacterial plasmid vector that is...
Suppose you digest the genomic DNA of a particular organism with
the restriction enzyme SauA. Then you ligate the resulting fragment
into a unique BamHI cloning site of a plasmid. The sequence of the
restriction sites and position of cleavage is shown below
Note: X and Y and their complementary bases Z and Y’
respectively can be any base (A,C, G, or T)
1) As you can see, ligation is possible because the two restriction
enzymes produce compatible sticky ends....
please help me with these questions
Lab 8 Extension Activity: Plasmid Mapping and Restriction Enzymes Mapping the Plasmid The first step in mapping a plasmid is to determine how many times a restriction site is found on that plasmid. Examine the results for plasmid 55 as an example. The data given in the following table are for the double digest using EcoRI and Pstl. Also, giving are the data for single digests by the individual enzymes. The numbers in the...
In your previous prac session you digested your POTC-A plasmid DNA with 3 enzyme mixes (AB and C). One mix contained the restriction endonuclease Kpnl alone, another contained both del and Not and another contained Sphi and Nhe, but you don't know which was which yet. The sites where these enzymes cut POTC-A are indicated on the plasmid map at the top of the page. Calculate the sizes of the DNA fragments that you would expect to see on your...
7. Explain the procedure for cloning DNA fragment into the plasmid PBR322 (shown on the right) (S pts.). The gene fragment of interest was obtained by digestion of chromosomal DNA with the restriction enzyme Sall and subsequent purification using agarose gel electrophoresis. Which antiblotic would you use in the final step of the cloning procedure, and Pst why? EcoR Sal Ampicillin Tetracycline resistanica(Ter Amp) PBR322 4,361 bp) Origin of replicatiorn (ori Pvull 8. Assume that your gene fragment from question...
1. You perform a restriction endonuclease assay on an uncharacterized DNA molecule, using Pstl, Sall, and Xhol. When you run the DNA on a 1% agarose gel, you obtain the following information regarding fragment number and length (all lengths are in bp): Pstl (P) 700 200 Sall(s) 600 300 Xhol (X) 500 350 50 Pstl + Sall (P+S) 600 200 100 Pstl + Xhol (P+X) 500 200 150 50 Sall + Xhol (S+X) 500 250 100 50 Solve the following...
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