Tutorial 1

Question 1

5’ end overhang

Answer 1

5’ A 3’

3’ TCCGTATT5’

Question 2

5’ end recessed

Answer 2

5’ AGGCATAA 3’

3’ T 5’

Question 3

3’ end overhang

Answer 3

5’ AGGCATAA 3’

3’ T 5’

Question 4

3’ end recessed

Answer 4

5’ A 3’

3’ TCCGTATT5’

Question 5

What is the efficiency patter of T4 PNK over different 5’ ends?

Answer 5

5′ overhangs efficiently, follow by blunt-ended and, then 5′ recessed ends. The two last ones are associated with a reduced efficiency

Question 6

Blunt end

Answer 6

5’ AGGC ATAA 3’

3’ TCCG TATT5’

Question 7

Canonical

Answer 7

Stick to a canon, what is usually expected to see.

For example DNA and RNA base pairing sticking to Watson-Crick rules of hybridization

Question 8

Non- Canonical

Answer 8

Does not stick to a canon, what is unexpected to see.
For example non-canonical base pairing occurs when nucleobases hydrogen bond, or base pair, to one another in schemes other than the standard Watson-Crick base pairs

Question 9

Why are OriR’s high in T and A?

Answer 9

A-T base pairs are held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs can be separated more readily at lower temperatures and allows the replication machinery room to come in and get busy making copies.

Question 10

What does it mean we have a “relax” Ori?

Answer 10

• Positively regulated by RNA or proteins
• No extra machinery needed
• High yield as a result

Question 11

What does it mean we have a “stringent” Ori?

Answer 11

• Negatively regulated by RNA or proteins
• Extra machinery from the host is needed
• Low yield as a result

Question 12

Are plasmid with the same original compatible or incompatible?

Answer 12

Incompatible because they will compete for the same machinery, creating an unstable and unpredictable environment

Question 13

What genes encodes for the Methyltransferases for Adenine and Cytosine?

Answer 13

The dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes that encode for the Dam and Dcm methyltransferases.

Question 14

What bases can suffer a methylation process?

Answer 14

Cytosine and Adenine

Question 15

Why is methylation important when doing enzyme digests?

Answer 15

Methylation should be considered when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular base in the recognition site is methylated

Question 16

Solution to undigested methylated restriction sites when cleaving DNA in the lab

Answer 16

Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by cloning your DNA into a dam–, dcm– strain of E. coli, such as dam–/dcm– Competent E. coli

Question 17

What is a MCS?

Answer 17

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids.

Question 18

What is the purpose of MCS?

Answer 18

The purpose of a MCS in a plasmid is to allow a piece of DNA to be inserted into that region.

Question 19

What are MCS also known as?

Answer 19

Polylinkers

Question 20

Star activity

Answer 20

is the relaxation or alteration of the specificity of a restriction enzyme mediated cleavage of DNA. The result is typically cleavage at non-canonigal recognition sites (which are recognition sites that differ from the normal, expected one), or sometimes complete loss of specificity.

Question 21

What substances can inhibit a reaction ( substrates)

Answer 21

Ethanol

Question 22

Why adding to much enzyme can inhibit a reaction

Answer 22

The problem is not the amount of enzyme (although in massive quantities it can induce a start activity), but mainly with the glycerol, as all enzymes are stored in this substance and using more than the advice quantity can inhibite the reaction.