BIOL 341 Lecture 2: Lab 2 - Restriction Analysis and PCR primer design - 2017W Term 1 (2)

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graycamel551

9 Dec 2017

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Lab 2: restriction analysis and pcr primer design. It is a good idea to confirm the identity of any plasmid that is prepped (grown in bacterial culture and isolated) or received from others, because tubes can get mislabeled, and plasmid preps may be compromised. Starting a project with a mislabeled plasmid or plasmid with defective structure can waste weeks of work! You will be working with two different plasmids. This week you will be carrying out restriction digests to identify which of these two plasmids you have received. The restriction fragments will be sized in the next lab through gel electrophoresis. This week you will also be planning the first steps in cloning the t7 promoter from pgem-t into pgfp-s12, and predicting the results you would see if your strategy is successful. Design and evaluate pcr primers that will amplify sequences and introduce new restriction sites.

Related Questions

Which of the following did Erwin Chargaff observe?

A)	Base composition changes as the organism ages.

B)	Base composition of DNA does not vary from one species toanother.

C)	In all species the number of adenines equals the number ofguanines.

D)	In all species A + T = G + C.

E)	DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)	the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)	the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)	the temperature at which double-stranded DNA is completelymelted.

D)	the temperature at which double-stranded DNA has become 50%single-stranded.

E)	the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)	Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)	Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)	Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)	A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)	Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)	Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)	Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)	When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)	None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)	DNA ligase

B)	Restriction endonuclease

C)	Reverse transcriptase

D)	DNA polymerase I

E)	Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)	Reverse transcriptase

B)	Alkaline phosphatase

C)	DNA ligase

D)	DNA polymerase I

E)	Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)	Alkaline phosphatase

B)	Reverse transcriptase

C)	Restriction endonuclease

D)	DNA ligase

E)	DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)	An origin of replication

B)	A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)	Unique restriction sites for insertion of DNA.

D)	Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)	All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)	Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)	cDNA libraries contain cDNAs made from mRNA.

C)	Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)	Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)	cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

dNTPs

B)	DNA containing the sequences to be amplified

C)	DNA ligase to connect the fragments together

D)	Primers complementary to each end of the sequence to beamplified

E)	Heat stable Taq polymerase

How can a fusion protein be formed?

A)	Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)	Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)	Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)	Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)	A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)	The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)	The reaction is only dependent on the presence of GFP and oneother cofactor.

C)	It is easily cloned and expressed in bacterial cells.

D)	The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)	It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)	Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)	Create a fusion with green fluorescent protein (GFP).

C)	Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)	A and B might work.

E)	A, B, and C might work.

F)	None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)	The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)	A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)	Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)	Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)	The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

3 Gm

300 Kb

3 Mb

3 Gb

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)	Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)	Eukaryotic genes often encode introns that are spliced outbefore translation.

C)	Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)	Eukaryotic genes are typically arranged in operons.

E)	Prokaryotic genes often encode exons that are spliced out beforetranslation

copperskunk824

Please answer all

Why does a template-dependent DNA polymerase require a primer to initiate DNA synthesis?

	DNA polymerase require 5' phosphate to add a new nucleotide
	DNA polymerase require 3' hydroxyl group to add a new nucleotide

DNA polymerase require energy by hydrolizing the primers

Which of the following enzymes is template independent?

	Reverse transcriptase
	Klenow fragment

	DNA polymerase
	Terminal deoxynucleotidyl transferase

RNA polymerase

The natural function of a DNA ligase enzyme is:

	Joining together DNA sequences with compatible cohesive ends from two different organisms to yield a recombinant DNA molecule
	Sealing single stranded nicks in double stranded DNA molecules

	Joining together two blunt ended fragments of DNA within a cell
	Joining together two single stranded DNA molecules

The natural function of 3' -> 5' exonuclease activity of a DNA polymerase is to:

	Remove the 5' end of the DNA strand that is being copied
	Remove damaged nucleotide from the template strand

	Remove nucleotides from the end of DNA to generate blunt ends
	Remove incorrect nucleotides from the newly synthesized DNA

Which technique is used to resolve the different sizes of DNA fragments?

	Gene cloning
	PCR

	DNA sequencing
	Gel electrophoresis

Which of the following vectors does not contain bacterial origin of replication (oriC)?

	plasmid
	cosmid

	bacterial artificial chromosome
	lambda vector

Which process of bacteriophage is not used for gene cloning?

	Concatemer formation
	Lytic cycle

	Lysogenic cycle
	viral packaging

What vector would be best suited for creating a contig of bovine (cattle) chromosome 10?

	lambda vector
	Cosmid vector

	P1 vector
	Plasmid

E. coli takes up plasmid DNA by which of the following methods?

	Transduction
	Transformation

	Conjugation
	Transfection

The following times and temperatures are an example of the steps for PCR. You can use the Figure to help you answer the following questions.

Why is the first step is carried out at 94Â°C?

	To degrade the template
	To denature the template

	To activate the polymerase
	To facilitate the primer binding

What happens in the reaction when the temperature shifts to 55Â°C during cycling?

	the DNA polymerase will carry out DNA synthesis by extending the annealed primers
	the DNA polymerase will finish DNA synthesis

	the primers anneal to the single-stranded regions of the DNA
	the primers anneal to the double-stranded regions of the DNA

During cycling, what occurs when the temperature is at 72Â°C?

	the primers anneal to the double-stranded regions of the DNA
	the DNA polymerase will carry out DNA synthesis by extending the annealed primers

	the primers anneal to the single-stranded regions of the DNA
	the DNA polymerase will finish DNA synthesis

You designed a set of primers to amplify 1 kb DNA with polymerase chain reaction. By which cycle of polymerase chain reaction, we would expect to see the first double stranded DNA with expected size?

	The end of cycle 1
	The end of cycle 2

	The end of cycle3
	The end of cycle 4

BIOL 341 Lecture 2: Lab 2 - Restriction Analysis and PCR primer design - 2017W Term 1 (2)

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