DNA cloning

Question 1

What is an insert?

Answer 1

Target DNA region that is incorporated into the vector.

Question 2

What are the two requirements for a vector?

Answer 2

1. DNA molecule that can integrate foreign DNA.
2. Can replicate it independently.

Question 3

What are the three genetic features of typical cloning vectors?

Answer 3

1. Origin of replication
2. Multiple cloning site / polylinker.
3. Selectable markers.

Question 4

The MCS allows for independent replication. True or false?

Answer 4

False.

Question 5

The origin of replication facilitates the addition of the insert. True or false?

Answer 5

False.

Question 6

Antibiotic resistance genes are a common selectable marker. True or false?

Answer 6

True.

Question 7

In the vector, there is a short stretch of DNA with many unique RE sites. What is it called?

Answer 7

MCS / polylinker.

Question 8

What is the really common plasmid vector called?

Answer 8

pUC19.

Question 9

What are the two selectable markers in the pUC19 vector?

Answer 9

1. Ampicillin resistance gene.
2. lacZ gene.

Question 10

Does transformation into host cells happen before or after ligation of the insert?

Answer 10

After.

Question 11

What enzyme carries out vector dephosphorylation?

Answer 11

Alkaline phosphatase.

Question 12

What type of enzyme carries out insert phosphorylation?

Answer 12

A kinase.

Question 13

What two things does vector dephosphorylation prevent?

Answer 13

1. Self-ligation.
2. Concatamerisation (different vectors bind).

Question 14

Insert phosphorylation is required for the activity of which enzyme?

Answer 14

T4 DNA ligase.

Question 15

What do we call cells that are more permeable to foreign DNA?

Answer 15

Competent.

Question 16

What chemical is used in chemical transformation?

Answer 16

Calcium chloride.

Question 17

What transformation technique uses short electric pulses?

Answer 17

Electroporation.

Question 18

If the insert is properly ligated, will lacZa be expressed?

Answer 18

No.

Question 19

We want a white phenotype in our cells. True or false?

Answer 19

True.`

Question 20

If beta-galactosidase is functional, do we have an insert-carrying vector?

Answer 20

No.

Question 21

What is the role of IPTG?

Answer 21

Induces the lac operon.

Question 22

If X-gal is not hydrolysed, what colour will the colony be?

Answer 22

White.

Question 23

Insertional inactivation will occur whether or not the insert is correct. True or false?

Answer 23

True.

Question 24

What is the main difference between a genomic and a cDNA library?

Answer 24

Genomic libraries contain all DNA, cDNA libraries contain only the expressed DNA.

Question 25

Lambda phages only infect E. coli. True or false?

Answer 25

True.

Question 26

What are the two structural components of a lambda phage?

Answer 26

A head (capsid) and a tail (sheath).

Question 27

What is a lytic cycle in viruses?

Answer 27

A replicative cycle where new phages are released via host lysis.

Question 28

What are three desirable features of lambda phages as vectors?

Answer 28

1. dsDNA (REs can work) 2. High infection rate. 3. Large regions of non-essential DNA.

Question 29

What range of insert sizes can a lambda phage take?

Answer 29

15-21 kb.

Question 30

What total amount of DNA must the lambda phage have to exist?

Answer 30

46-52 kb.

Question 31

What is the lysate?

Answer 31

The culture of lysed bacteria and recombinant lambda phages.

Question 32

What do circular plaques on the bacterial lawn tell us?

Answer 32

E. coli cells have died.

Question 33

Each plaque represents a unique phage. True or false?

Answer 33

True.

Question 34

What is used to 'copy' the bacterial lawn?

Answer 34

A membrane/filter.

Question 35

Is the membrane treated with acidic or alkaline solution? Why?

Answer 35

Alkaline solution, to release DNA and denature it.

Question 36

DNA probes bind with dsDNA to identify the target insert. True or false?

Answer 36

False, ssDNA.

Question 37

Two possible ways to produce sticky ends for cDNA libraries?

Answer 37

1. Restriction enzymes. 2. Ligation of restriction site linkers.

Question 38

What enzyme is required for the first step of cDNA library creation?

Answer 38

Reverse-transcriptase.

Question 39

Bacteriophages are lysed on the membrane during lambda phage DNA cloning. True or false?

Answer 39

True.