Molecular - Cloning

Question 1

Traits of a good cloning vector

Answer 1

Multiple cloning sites; antibiotic resistance (Amp or Kan) or GFP, origin of replication

Question 2

What are clones good for?

Answer 2

Looking at genomes - each plasmid will get one piece, and all the plasmids (10,000s) represent the genome

Question 3

cDNA library

Answer 3

It is a snapshot of expression, made from mRNA

Question 4

How do we know if a colony contains gene of interest?

Answer 4

blue/white screening: clone has ßgal gene, treat with Xgal and IPTG - when ßgal is active it cleaves Xgal and creates blue. If clone contains inserts, then ßgal doesn’t work and colony is white

Question 5

What is important to consider with regards to the multiple cloning site?

Answer 5

Ensure that the rest of the plasmid has no cut sites!

Question 6

What is Xgal?

Answer 6

A chlorinated disaccharide which is cleaved by ßgal and forms blue precipitate

Question 7

Where would you put ßgal in cloning vector?

Answer 7

What the multiple cloning site to be in the middle of ßgal. this prevents its function when an insert is present and the colony will be white.

Question 8

IPTG

Answer 8

Induces the ßgal gene in the plamid (triggers transcription of lac operon)

Question 9

Role of alkaline phosphatase in cloning

Answer 9

Prevents plasmid ends from reannealing. It clips the terminal (P) on the ends so it doesn’t close up again. Only the insert can fall in there and close it up. The increases the number of white colonies.

Question 10

Why would you include certain promoters in a plasmid?

Answer 10

Then the plasmid will be activated in tissues, ex. use actin promoter because it’s very common. Can also make it more specific.

Question 11

Why would you include eukaryote and prokaryote promoters in a plasmid?

Answer 11

Makes it easier to transition between different systems, such as animal, invertebrate, and bacterial