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Molecular Biology

Robert F. Weaver

Chapter 4

Molecular Cloning Methods - all with Video Answers

Educators

Chapter Questions

04:42

Problem 1

Consulting Table 4.1, determine the length and the nature ( $5^{\prime}$ or $3^{\prime}$ ) of the overhang (if any) created by the following restriction endonucleases:
a. $A l u \mathrm{I}$
b. $B g l \Pi$
c. ClaI
d. $K p n I$
e. $M b o l$
f. PvuI
g. NotI

Jennifer Stoner
Jennifer Stoner
Numerade Educator
04:06

Problem 1

Here is the amino acid sequence of part of a hypothetical gene you want to clone:
Pro-Arg-Tyr-Met-Cys-Trp-Ile-Leu-Met-Ser
a. What sequence of five amino acids would give a 14 -mer probe with the least degeneracy for probing a library to find your gene of interest? Notice that you do not use the last base in the fifth codon because of its degeneracy.
b. How many different 14 -mers would you have to make in order to be sure that your probe matches the corresponding sequence in your cloned gene perfectly?
c. If you started your probe one amino acid to the left of the one you chose in (a), how many different 14 -mers would you have to make? Use the genetic code to determine degeneracy.

Sana Riaz
Sana Riaz
Numerade Educator
03:37

Problem 2

Why does one need to attach DNAs to vectors to clone them?

Sana Riaz
Sana Riaz
Numerade Educator
01:34

Problem 2

You are cloning the genome of a new DNA virus into pUC18. You plate out your transformants on ampicillin plates containing X-gal and pick one blue colony and one white colony. When you check the size of the inserts in each plasmid (blue and white), you are surprised to find that the plasmid from the blue colony contains a very small insert of approximately $60 \mathrm{bp}$, while the plasmid from the white colony does not appear to contain any insert at all. Explain these results.

Jessica Honkomp
Jessica Honkomp
Numerade Educator
01:39

Problem 3

Describe the process of cloning a DNA fragment into the BamHI and PstI sites of the vector pUC18. How would you screen for clones that contain an insert?

Rashmi Sinha
Rashmi Sinha
Numerade Educator
05:17

Problem 4

Describe the process of cloning a DNA fragment into the EcoRI site of the Charon 4 vector.

Eric Tran
Eric Tran
Numerade Educator
00:10

Problem 5

You want to clone a $1-\mathrm{kb}$ cDNA. Which vectors discussed in this chapter would be appropriate to use? Which would be inappropriate? Why?

Sam Limsuwannarot
Sam Limsuwannarot
Numerade Educator
01:54

Problem 6

You want to make a genomic library with DNA fragments averaging about $45 \mathrm{~kb}$ in length. Which vector discussed in this chapter would be most appropriate to use? Why?

Kendrick Buford
Kendrick Buford
Numerade Educator
02:29

Problem 7

You want to make a library with DNA fragments averaging over $100 \mathrm{~kb}$ in length. Which vectors discussed in this chapter would be most appropriate to use? Why?

Sana Riaz
Sana Riaz
Numerade Educator
02:28

Problem 8

You have constructed a cDNA library in a phagemid vector. Describe how you would screen the library for a particular gene of interest. Describe methods using oligonucleotide and antibody probes.

Ummatul Choudary
Ummatul Choudary
Numerade Educator
03:43

Problem 9

How would you obtain single-stranded cloned DNAs from an M13 phage vector? From a phagemid vector?

Deborah Greenspan
Deborah Greenspan
Numerade Educator
03:15

Problem 10

Diagram a method for creating a cDNA library.

Dennis Howard
Dennis Howard
Numerade Educator
01:55

Problem 11

Diagram the process of nick translation.

Lottie Adams
Lottie Adams
Numerade Educator
04:22

Problem 12

Outline the polymerase chain reaction (PCR) method for amplifying a given stretch of DNA.

Ricajoy Montero
Ricajoy Montero
Numerade Educator
02:28

Problem 13

What is the difference between reverse transcriptase PCR (RT-PCR) and standard PCR? For what purpose would you use RT-PCR?

Dennis Howard
Dennis Howard
Numerade Educator
00:38

Problem 14

Describe the use of a vector that produces fusion proteins with oligohistidine at one end. Show the protein purification scheme to illustrate the advantage of the oligohistidine tag.

Rachel Vallejo
Rachel Vallejo
Numerade Educator

Problem 15

What is the difference between a $\lambda$ insertion vector such as $\lambda g t 11$ and a $\lambda$ replacement vector? What is the advantage of each?

Check back soon!
03:42

Problem 16

Describe the use of a baculovirus system for expressing a cloned gene. What advantages over a bacterial expression system does the baculovirus system offer?

Khalida Dawar
Khalida Dawar
Numerade Educator
03:06

Problem 17

What kind of vector would you use to insert a transgene into a plant such as tobacco? Diagram the process you would use.

Dennis Howard
Dennis Howard
Numerade Educator