Workshop 2

Question 1

What cut does HindII produce ? - and PstI ?

Answer 1

HindII : GTC/GAC (blunt)

PstI : CTGCA/G (4nt long3’ overhangs)

Question 2

What are isoschizomers ?

Answer 2

Isoschizomers = restriction enzymes that recognise the same sequence but originate from different species.
For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.

Question 3

What are neoschizomers ?

Answer 3

Neoschizomers = restriction enzymes that have the same recognition site but generate different ends

Question 4

What are isocaudomers ?

Answer 4

Isocodaumers = restriction enzymes that produce the same nucleotide extensions (overhangs).

Question 5

Which fragment length would a class II restriction enzyme with a recognition site of 4 bp generate if the template is a random sequence ?

Answer 5

1/4 * 1/4 * 1/4 *1/4= 1/256
Statistically the site would be present every 256 bp,
consequently the expected length would be 256 bp

Question 6

What is star activity ?

Answer 6

Star activity: Cutting of a restriction endonuclease at sites resembling the recognition sequence (e.g. GAATTC => AATTC)

Question 7

What are the factors favoring star activity ?

Answer 7

• increased concentration of glycerol best < 5% some enzymes (EcoRI, BamHI < 2%)
• pH too low/high (different buffer sets)
• ionic strength too low/high (different buffer sets)
• divalent cofactors (Mn2+, Cu2+, Co2+, Zn2+)
• organic solvents (Ethanol, DMSO, Dimethylformamide, volume excluder (e. g. ethylene glycol)
• prolonged reaction time
• increased amount of enzyme

Question 8

What specific DNA methylations can be observed in E. Coli K13 strains ?
What effect does this have ?

Answer 8

E. coli K12 strains are normally dam+ and dcm+ which results in inhibition of certain restriction enzymes.

• dam: methylation results in a N6-methyladenine (m6A) in the following sequence 5’-GATC-3’ (inhibits MboI (/GATC) activity)
• dcm: methylation results in a 5-methylcytosine (m5C) on the second C in 5’-CCWGG-3’ (inhibits SexA1 (A/CCWGGT) activity)

Question 9

What is the basic unit of agarose ?

Answer 9

The basic unit of agarose is a disaccharide called agarobiose (1,3-linked ß-D-galactopyranoe & 1,4-linked 3,6-anhydro-alpha-L galactopyranose ).

Question 10

Describe what happens during agarose gelation.

Answer 10

Gelation = shift from random coil in solution
Double helix (initial stages)
Bundles of double helix (late stage)
Pore sizes = 100 - 300 nm

Question 11

Define electrophoresis.

Answer 11

Electrophoresis is defined as the movement of ion and charged macromolecules through a medium when an electric current is applied.

Question 12

What are the primary stationary media used in electrophoresis for macromolecules ?

Answer 12

Agarose and polyacrylamide.

Question 13

What does the seperation of macromolecules in an agarose gel depend on ?

Answer 13

Charge, size and structure (topology).

Question 14

State Ohm’s law.

What does resistance depend on in gel electrophoresis ?

Answer 14

V = IR

Buffers, type of matrix and configuration (horizontal, vertical) & volume of gel/buffer.

Question 15

How can we measure the heat produced by the electrophoresis ?

Answer 15

Measure of produced heat: Power (W) = I * V = I^2 * R also known as Joule’s law

Question 16

What happens to water during gel electrophoresis ?

What happen at the cathode/anode ?

Answer 16

It is electrolyzed.
Anode (+): oxygen and protons -> acidic
Cathode (-): Hydrogen and hydroxyl ions -> basic

Question 17

What are the 2 functions of the buffer in electrophoresis ?

Answer 17

Buffer –> 2 functions:

• transport charge
• keep molecules uniformly charged throughout separation process

Question 18

What are the compositions of TAE and TBE ?

Answer 18

TAE - 40 mM Tris-acetate, 1 mM EDTA

TBE - 89 mM Tris-borate, 2 mM EDTA

Question 19

What is electroendosmosis (EEO) ?

Answer 19

EEO is the movement of liquid in a porous material due to an applied electric field.

Question 20

How is agarose charged ?
What effect does this have ?
What can we therefore say about the net water flow ?

Answer 20

Agarose mainly neutral but some anionic residues
(sulfate, pyruvate,…) –> move towards the anode (+)
Hydrated counterions moves towards the cathode (-), with some neutral molecules dragged along
This means that the net flow of water i the gel is towards the cathode.

Question 21

What effect does high EEO have on migration speed ?

Answer 21

When EEO is high migration of DNA is retarded.

Question 22

When should TAE buffer be used ?

Answer 22

• when DNA is to be recovered
• for electrophoresis of large (>10 kb) DNA
• low buffer capacity – recirculation

Question 23

When should TBE buffer be used ?

Answer 23

• for electrophoresis of small (<1 kb) DNA
• increased resolution of small (1.5 kb) DNA
• high buffer capacity – decreased DNA mobility

Question 24

What is the consequence of excessive salt concentration on gel electrophoresis results ?
What if there is no salt ?
What is a goof rule of thumb for [salt] in 0.8% agarose ?

Answer 24

Too high salt = appearance of smears + fragment migrate slower than they normally should
No salt = apperance of smears
Good rule of thumb : [NaCl] = 0.25M for 0.8% agarose