21. E. Coli transformation & Selection systems for Cloning

Question 1

define competent cells and how they are made

Answer 1

cells that are more likely to incorporate foreign DNA because their cell walls are altered so DNA can pass through more easily.

Using calcium chloride and heat shock makes competent cells

Question 2

how are competenet cells preparened and tansformed?

7 steps

Answer 2

1. cells in log phase (exponential growth) are centrifuged
2. suspend centrifuged cells in CaCl2
3. place on ice for 20 minutes
4. add DNA, DNA binds to Ca
5. heat shock
6. DNA uptake by cell
7. plate on media and transformants grow

look carefully at picture!!

Question 3

what is transformation?

Answer 3

when DNA is taken up by the cell from its surroundings

Question 4

what is on the agar plates when plating the transformed cells and why

Answer 4

there is antibiotic on the agar plate. Because the transformed cell will have an antibitotic resistance gene in it so that only transformed cells will survive

Question 5

what are the three types of cells that will be grown on the media?

Answer 5

1. dead cells (didnt take up the plasmid)
2. live cells without recombinant plasmid (have a plasmid but not the gene of interest)
3. live cells that are recombinants (have a plasmid and the gene of interest)

Question 6

what is random selection, what are the 5 steps for it?

Answer 6

random selection is when the colonies on the agar plate are picked and grown in a liquid culture,

centrifuged to collect bacteria

then isolated to extract the plasmids.

this is then digested with restriction enzymes (cut up)

and run on gel electrophoresis.

Question 7

why is random selection done?

Answer 7

because the colonies that live on the plate dont all have the gene of interest in their plasmid. need to differentiate them

Question 8

what are the two methods of plasmid analysis?

Answer 8

1. plasmid restriction digests
2. PCR analysis of plasmid DNA or cells

note- the analysis occurs after picking, centrifuging and isolating plasmids. these are two seperate ways to do it

Question 9

how does insertional inactivation work?

Answer 9

the cloning site is within two antibitotic resistant genes.

When DNA (with gene of interest) is inserted, it inactivates one of the antibiotic resisitant genes. can select for the host cell recombinant plasmids using other antibiotic

Question 10

what is replica plating?

Answer 10

when another agar plate containing different nutrients (ie a different antibiotic on it) are incoluclated from the original medium.

Question 11

why is replica plating used in negative selection?

Answer 11

This is used to compare which are the non recombinants (will grow on both antibiotics) and recombinants (will only grow on first antibiotic plate because the recombinant gene will be in the middle of the other recombinant gene)

look at image carefully!!

Question 12

how does blue/white screening for recombinants work?

Answer 12

the colonies are grown on X-gal plates. if the X-gal is hydrolysed by B-galactosidase, it makes a blue pigment.

the gene of interest on the plasmid is inserted where the B-galactoside gene is, therefore the gene is inactivated and a white pigment is made and X-gal is not metabolised

Blue colonies = non recombinants

white colonies = recombinants