Practical A Flashcards
EcoR1
- 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
Alu1
- Mainly not methylation sensitive
- Optimal - 37C
- Sensitive to heat
- Isolated from Arthrobacter luteus
- Blunt ends: recognises AGvCT site and cuts straight through double
Bgl2
- 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
Why is agarose used?
Long unbranched chain carbohydrate without cross-links, allows large pores +macromolecules
Why is glycerol used?
Makes samples dense and makes them sink into agarose well
Bromophenol blue is used because
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)
Why are gloves used?
TBE contains ethidium bromide - potential carcinogen
Overlapping bands are due to
Incomplete separation
Weight size markers are
A mixture of nucleotides of known sizes, used for comparison with unknown bands
Migration pattern of single stranded nucleic acid
Tend to fold up into complex shapes and migrate in a complicated manner depending on tertiary structure
When plotting calibration graph for gel electrophoresis Log10 base pairs is on which axis?
X
The distance the band travels is inversely proportional to
Size of the molecule
