Rodgers 2-28

Question 1

lac I gene

Answer 1

lac repressor- prevents expression of the protein until the cells are growing very strongly

Question 2

lac repressor

Answer 2

usually always expressed by the bacteria. recognition site can be in both the lac operon (blocks the T7 RNA polymerase expression controlled by lac operon), and in the T7 RNA polymerase binding site

Question 3

What additional safeguard is put in place to prevent T7 polymerase from being expressed before desired induction?

Answer 3

T7 lysozyme constitutively expressed- so T7 polymerase expression levels must exceed T7 lysozyme levels to be active in the cell

Question 4

IPTG

Answer 4

chemical that mimics lactose to turn off lac I gene, discontinue lac repressor expression

Question 5

How are bacteria protected against the action of their own Restriction Enzymes?

Answer 5

Methylation of their DNA

Question 6

What structure do RE’s have

Answer 6

homodimer

Question 7

What mechanism do RE’s use to home in on cleavage sequences

Answer 7

they bind non-specifically and slide along the DNA, then undergo conformation change to bind tightly and cleave

Question 8

How are RE’s named?

Answer 8

First letter genus, second two letters species, number represents the strain or serotype

Question 9

What is a feature of all RE cleavage recognition sites?

Answer 9

palindromic

Question 10

How long are RE recognition sites

Answer 10

4-8 bp’s

Question 11

Why are types II and V RE’s preferred over the other RE’s

Answer 11

The others usually cut non-specifically or at a distance from the recognitions sites

Question 12

Summary of the chemical mechanism used by RE’s

Answer 12

conserved Asp residues in the active site coordinate divalent ion Mg to hold H2O in place to attack the backbone

Question 13

What enzyme is critical to insertion into the vector after cleavage by the RE?

Answer 13

DNA ligase

Question 14

What is the natural system bacteria have for uptaking DNA

Answer 14

plasmids enter through channels that chew up one of the strands, so only single strand DNA enters the cell (associated with the pilus proteins)

Question 15

How are cells made competent by heat shock

Answer 15

they are chemically treated with cold salty water (disrupts the membrane), added to glycerol, and flash frozen before administering heat shock while incubated with plasmid. Chill on rich medium and spread over selectable antibiotic.

Question 16

How are cells made competent by electroporation?

Answer 16

they are given several washes in cold water (to tone down ion content so they don’t fry), then placed in glycerol and flash frozen, pulse with electricity, place in chilled rich media, and spread over selectable antibiotic

Question 17

How are RNA’s amplified to make cDNA’s

Answer 17

Use poly-A as the rev primer and random hexamers

Question 18

Preferred melting point and length of primers

Answer 18

25-45 bps, >72 C

Question 19

What should one remember about GC content in primers?

Answer 19

They should constitute ~50-60% and the 3’ end should be a G or a C to prevent ‘breathing’ but should not end in 3 or more C/G’s to prevent preferential binding to GC rich regions

Question 20

At what temperature does Taq Polymerase activate?

Answer 20

72C