Practical A

Question 1

EcoR1

Answer 1

• Isolated from E.coli
• RY13 particular strain from where it’s isolated from
• Nucleic acid recognition seq. = GAATTC but cuts so 4 nucleotide sticky ends with 5’end overhangs (AATT)
• Homodimer
• Conditions: low salt conc., high glycerol conc. , excessive amounts of enzyme present in the reaction, high pH + organic solvent contamination

Question 2

Alu1

Answer 2

• Mainly not methylation sensitive
• Optimal - 37C
• Sensitive to heat
• Isolated from Arthrobacter luteus
• Blunt ends: recognises AGvCT site and cuts straight through double

Question 3

Bgl2

Answer 3

• Isolated from certain strains of Bacillos Globigai
• Consists of two identical subunits that form a homodimer around the DNA double helix
• Each monomer is 273 aa long and symmetrically cleaves AGATCT at the phosphodiester bond between first A + G nucleotides on both strands
• Doesn’t require ATP for enzymatic function
• Requires association with a divalent metal cation e.g. Mg2+
• Undergoes conformational change upon dimerisation but overall mech. remains consistent with outer type 2 restriction enzymes

Question 4

Why is agarose used?

Answer 4

Long unbranched chain carbohydrate without cross-links, allows large pores +macromolecules

Question 5

Why is glycerol used?

Answer 5

Makes samples dense and makes them sink into agarose well

Question 6

Bromophenol blue is used because

Answer 6

It runs ahead of the smallest fragments + indicates when electrophoresis is complete. Experiment can’t run for too long or buffering capacity can be exhausted and physical properties of DNA could be impaired (-ve charge)

Question 7

Why are gloves used?

Answer 7

TBE contains ethidium bromide - potential carcinogen

Question 8

Overlapping bands are due to

Answer 8

Incomplete separation

Question 9

Weight size markers are

Answer 9

A mixture of nucleotides of known sizes, used for comparison with unknown bands

Question 10

Migration pattern of single stranded nucleic acid

Answer 10

Tend to fold up into complex shapes and migrate in a complicated manner depending on tertiary structure

Question 11

When plotting calibration graph for gel electrophoresis Log10 base pairs is on which axis?

Answer 11

X

Question 12

The distance the band travels is inversely proportional to

Answer 12

Size of the molecule