Lecture 23

Question 1

How were restriction enzymes discovered?

Answer 1

Using bacteriophage that formed plaques.

Question 2

What does ‘pfus’ stand for?

Answer 2

Plaque forming units (every plaque can make a particle).

Question 3

How does methylation affect restriction enzyme activity?

Answer 3

It methylates the DNA so that the RE can’t cut it. This is a bacterial defence against phage.

Question 4

What size DNA fragments does the acrylamide gel analyse?

Answer 4

1bp - 1kb (ssDNA)

Question 5

What size DNA fragments does the agarose gel analyse?

Answer 5

0.5 - 20kb (plasmid clones)

Question 6

What size DNA fragments do pulse field gels analyse?

Answer 6

50kb - 10Mb (Large DNA Frags)

Question 7

What is the frequency of a 6 cutter cutting the DNA fragment?

Answer 7

(1/4)^6 = 1/4 kb

Question 8

What does Ligase do?

Answer 8

Reconnects phosphodiester bonds in the DNA

Question 9

What does Ligase need in order to anneal the DNA fragments?

Answer 9

5’ phosphate, 3’ OH
ATP
Mg2+

Question 10

What are the two types of DNA Transformation?

Answer 10

Chemical treatment and electroporation.

Question 11

Which type of DNA transformation has no size limit and is easy to perform?

Answer 11

electroporation.

Question 12

What is the copy number of the pUC plasmid?

Answer 12

200 per cell.

Question 13

What synthetic DNA is present on a plasmid within the lac-alpha DNA?

Answer 13

A multiple cloning site.