Lecture 5

Question 1

Site directed mutagenesis general plan

Answer 1

Create primer with new mutation

Amplify plasmid 1x

Dpn1 to digest methylated dna

Transformation into bacteria

Screening

Question 2

Why is ligase not needed prior to transformation?

Answer 2

Bacteria will repair the nick

Question 3

Is transformation inefficient?

Answer 3

Yes

Question 4

Selection example

Answer 4

Ampicillin resistance selection

Only cells that are resistant remain

Question 5

Screening example

Answer 5

Attempting to only have cloned vectors present

Utilizes effects of MCS disruption inherent to cloning into MCS

Blue/ white screening

Question 6

Screening vs selection

Answer 6

Selection = looking for successful transformations = all vectors contain resistant gene separate from MCS = selection marker

Screening = looking for transformations with desired clone

Question 7

Self ligate means

Answer 7

Bring ends together after restriction enzyme snipping just by chance overlap rather than by primers designed to overlap

Question 8

Bacteria can only uptake circular plasmids?

Answer 8

True

Vector with insert

Or

Self ligated vector

Or

Original plasmid

Question 9

Possible successful transformations

Question 10

Blue/ white screen details

Answer 10

Cloning within MCS creates gene disruption

Intact lacZ gene = blue = wt

Interrupted lacZ gene = white = mutant

Question 11

Why does lacZ result in different colors in different scenarios?

Answer 11

?

Question 12

Should empty vectors have pcr product?

Answer 12

Primers will not work on them