Gene Cloning Tools

Question 1

Describe how the vector DNA had to be prepared for ligation?

Answer 1

Purify with alkaline lysis, digest with restriction enzymes and treat with alkaline phosphatase to remove the 5’ phosphates.

Question 2

Describe how the target DNA had to be prepared for ligation?

Answer 2

Purify, PCR amplify with designed primers and digest with restriction enzymes.

Question 3

What occurs after ligation of the vector and target DNA?

Answer 3

The ligation mix is transformed into competent E.coli, plate into an agar plate with an antibiotic, screen colonies to identify those with recombinant plasmid- colony PCR or plasmid isolation and restriction digest.

Question 4

How many DNA molecules dies each cell take up during transformation?

Answer 4

1 per cell

Question 5

How is cDNA made?

Answer 5

From mRNA using reverse transcriptase and a primer.

Question 6

What primers are used for cDNA synthesis?

Answer 6

For first strand synthesis usually an oligo dT and for second strand synthesis usually a self-priming or specific primer plus DNA polymerase

Question 7

What is PCR?

Answer 7

Polymerase chain reaction: a method of DNA amplification

Question 8

How many cycles of thermal cycling does PCR require?

Answer 8

25-40

Question 9

Describe a PCR thermal cycle?

Answer 9

90 oC initial denaturation, 55 oC anneal primers, 72 oC DNA synthesis (extension).

Question 10

What enzymes are required for PCR?

Answer 10

Thermostable DNA polymerases

Question 11

Give an example of some thermostable DNA polymerases?

Answer 11

Taq: 5’ -3’ exonuclease and DNA synthesis

Kod, Pfu: 5’-3’ DNA synthesis and 3’-5’ proof-reading exonuclease.

Question 12

During PCR how does the DNA get amplified in each step?

Answer 12

Exponentially

Question 13

What has to be considered when designing a primer?

Answer 13

Length, GC content, melting temperature, complimentarity, polypyrimidine/polypurine sequences.

Question 14

What length should a suitable primer be?

Answer 14

~20 nucleotides long

Question 15

What GC content should a suitable primer have?

Answer 15

~50%

Question 16

What melting temperature should a suitable primer have?

Answer 16

Similar for both, > 55 oC

Question 17

Why is it important to assess complimentarity of primers?

Answer 17

As you do not want them to be complementary to one and other.

Question 18

Why is it important to assess polypyrimidine and polypurine content of primers?

Answer 18

As you do not want stretched of either.

Question 19

What can you do to the 5’ end of primers?

Answer 19

Add sequences.

Question 20

What are some common cloning vectors?

Answer 20

Plasmids, viruses/bacteriophage, cosmids and phagemids

Question 21

What is a cosmid a combination of?

Answer 21

Plasmid and bacteriophage lambda

Question 22

What is a phagemid a combination of?

Answer 22

Plasmid and bacteriophage M13

Question 23

What do vectors used for cloning usually have?

Answer 23

Multiple cloning sites, promoter, origin of replication, antibiotic resistance, a method (eg. Tag) for purification.

Question 24

What are restriction sites?

Answer 24

6 nucleotide stretches of DNA which are recognised by restriction enzymes and ensure cleavage occurs on both the vector and target DNA in order for ligation.

Question 25

How is agarose gel electrophoresis used to analyse DNA fragments?

Answer 25

DNA and RNA have a net negative charge due to phosphate groups so they are attracted towards the anode. Larger molecules move slower through the matrix and smaller molecules faster.

Question 26

What sort of ends can restriction enzymes leave?

Answer 26

Blunt or sticky (overhang)

Question 27

Before transformation what are E.coli cells treated with and why?

Answer 27

CaCl2 or RbCl2 to disrupt their cell walls

Question 28

How are E.coli cells encouraged encouraged to take up the DNA?

Answer 28

By heat shock

Question 29

Why when plated on agar with antibiotics do only clones that contain the vector grow?

Answer 29

As they encode antibiotic resistance.

Question 30

What are some key molecular biology tools?

Answer 30

Vectors
Agarose gels
Restriction enzymes
Modifying enzymes
Ligases
Polymerases
Synthetic DNA
Polymerase chain reaction (PCR)