Lecture 4

Question 1

Forward and reverse primers should have similar Tm

Determined by?

Answer 1

G/C content

This is because GC has three H bonds = more stable = higher Tm

Question 2

If Tm too low, to get Tm of 50-60
Extend primer length

Why?

Answer 2

More h bonding total

Question 3

Should 3’ end of primer end in AT or GC?

Answer 3

GC

Stronger bond to polymerase

Question 4

Annealing temp formula

Answer 4

Tm-5 degrees

Question 5

Important conversion factor for PCR timing

Answer 5

Extension time for polymerase : Blank seconds per kb DNA

For phusion polymerase: 15 seconds per kb DNA

Question 6

Steps to GA cloning

Answer 6

1. Choose vector
2. Amplify vector and insert via pcr
3. Digest products with Dpni and clean up
4. Assemble vector / insert via Gibson assembly
5. Transformation into cells
6. Screen for successful clones

Question 7

Is the ori species specific?

Answer 7

Yes

Question 8

What does Ori control?

Answer 8

Copy number = number of plasmids per cell

Question 9

Induced vs constitutive promoter

Answer 9

Induced = turned on

Constitutive = always on

Question 10

Dpn1

Answer 10

Digestive enzyme
Only attacks methylated DNA = non pcr DNA

Question 11

Traditional cloning relationship with restriction enzyme

Answer 11

Primer overhangs were restriction enzyme sites = sticky overhangs
TTAA of insert overlaps with AATT of vector = sense vs antisense strands

Trick is that each insert / vector end needs unique sticky overhang

Question 12

Restriction enzymes recognize blank

Answer 12

Palindromic 6 base sequences