Hi please help with this its very important for my grade worth 25 points of my grade:
The AT-vector containing the rac PCR sequences AND the expression vector DNA to be used for cloning are digested with XhoI and EcoR1. The fragments were then mixed and joined together before transformation.
(a) (4 points) â Why were two different restriction enzymes used?
(b) (4 points) â If the cloning had been done with only a single restriction enzyme (both PCR primers contained the same restriction enzyme site and the vector was digested with only that one enzyme [for example EcoR1]), what additional step would have to be done to ensure that the correct clone was obtained?
(c) If the vector DNA to be used for cloning is digested with BamH1 and the TA-vetor/rac DNA used for cloning is digested with Sau3A, would using these two enzymes as described work for a cloning experiment? Explain
(d) (4 points) - What is the name of the enzyme used to join the ends of the target and vector DNA molecules together?
(e) (4 points) - What would happen during the ligation reaction if the restriction enzyme was not heat inactivated before the ligation reaction?
(f) (4 points) - Why was it necessary to treat the vector with phosphatase?
Hi please help with this its very important for my grade worth 25 points of my grade:
The AT-vector containing the rac PCR sequences AND the expression vector DNA to be used for cloning are digested with XhoI and EcoR1. The fragments were then mixed and joined together before transformation.
(a) (4 points) â Why were two different restriction enzymes used?
(b) (4 points) â If the cloning had been done with only a single restriction enzyme (both PCR primers contained the same restriction enzyme site and the vector was digested with only that one enzyme [for example EcoR1]), what additional step would have to be done to ensure that the correct clone was obtained?
(c) If the vector DNA to be used for cloning is digested with BamH1 and the TA-vetor/rac DNA used for cloning is digested with Sau3A, would using these two enzymes as described work for a cloning experiment? Explain
(d) (4 points) - What is the name of the enzyme used to join the ends of the target and vector DNA molecules together?
(e) (4 points) - What would happen during the ligation reaction if the restriction enzyme was not heat inactivated before the ligation reaction?
(f) (4 points) - Why was it necessary to treat the vector with phosphatase?
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View/perform/read ALL THREE of the following prior to answeringthe questions.
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)
http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)
http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)
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From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA
| IV, V, I, II, III |
| III, II, IV, V, I |
| III, IV, V, I, II |
| II, III, V, IV, I |
| I, II, IV, III, V |
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Plasmids (or vectors) are important in biotechnology becausethey are
| a vehicle for the insertion of recombinant DNA intobacteria. |
| surfaces for respiratory processes in bacteria. |
| recognition sites on recombinant DNA strands. |
| surfaces for protein synthesis in eukaryotic recombinants. |
| proviruses incorporated into the host DNA |
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Plasmids are put into bacterial cells by
| restriction enzymes |
| DNA ligase |
| binding of cohesive sticky ends |
| transformation |
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Restriction enzymes usually
| cut donor DNA evenly so smooth edges result |
| cut donor DNA but do not affect plasmids |
| make staggered cuts at specific sequences in DNA in both donorDNA and plasmid |
| are used in ligating plasmids into bacterial host cells |
| more than one of the above |
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After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.
| Restriction enzyme |
| DNA Ligase |
| Reverse transcriptase |
| DNA polymerase |
| Helicase |
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It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?
| All organisms have ribosomes. |
| All organisms have the same genetic code. |
| All organisms are made up of cells. |
| All organisms have similar nuclei. |
| All organisms have transfer RNA. |
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Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to
| cut the plasmid with restriction enzyme X and insert thefragments cut with Y into the plasmid. |
| cut the plasmid with enzyme X and then insert the gene into theplasmid. |
| cut the DNA again with restriction enzyme Y and insert thesefragments into the plasmid cut with the same enzyme. |
| cut the plasmid twice with restriction enzyme Y and ligate thetwo fragments onto the ends of the human DNA fragments cut withrestriction enzyme X. |
| insert the fragments cut with X directly into the plasmidwithout cutting the plasmid. |
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Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?
| They are used to make proteins using a cloned gene. |
| They contain a promotor. |
| They are the first plasmid type used to clone a gene. |
| They contain a terminator. |
| More than one of the above is false. |
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What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?
| If the eukaryotic gene contains introns the bacteria will notremove them and the resulting amino acid sequence will be differentthat that made by a eukaryote. |
| The bacteria may not fold the protein correctly. |
| The bacteria may degrade the protein. |
| All of the above are potential problems. |
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Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.
| True |
| False |
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Question 111 pts
Gene cloning is used to do all of the following except
| Make insulin |
| Making genetically identical animals (e.g. Dolly thesheep) |
| Make vaccines |
| Perform Gene Therapy |
| Making genetically engineered plants |
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