Workshop 2 Flashcards
What cut does HindII produce ? - and PstI ?
HindII : GTC/GAC (blunt)
PstI : CTGCA/G (4nt long3’ overhangs)
What are isoschizomers ?
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.
What are neoschizomers ?
Neoschizomers = restriction enzymes that have the same recognition site but generate different ends
What are isocaudomers ?
Isocodaumers = restriction enzymes that produce the same nucleotide extensions (overhangs).
Which fragment length would a class II restriction enzyme with a recognition site of 4 bp generate if the template is a random sequence ?
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
What is star activity ?
Star activity: Cutting of a restriction endonuclease at sites resembling the recognition sequence (e.g. GAATTC => AATTC)
What are the factors favoring star activity ?
- 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
What specific DNA methylations can be observed in E. Coli K13 strains ?
What effect does this have ?
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)
What is the basic unit of agarose ?
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 ).
Describe what happens during agarose gelation.
Gelation = shift from random coil in solution Double helix (initial stages) Bundles of double helix (late stage) Pore sizes = 100 - 300 nm
Define electrophoresis.
Electrophoresis is defined as the movement of ion and charged macromolecules through a medium when an electric current is applied.
What are the primary stationary media used in electrophoresis for macromolecules ?
Agarose and polyacrylamide.
What does the seperation of macromolecules in an agarose gel depend on ?
Charge, size and structure (topology).
State Ohm’s law.
What does resistance depend on in gel electrophoresis ?
V = IR
Buffers, type of matrix and configuration (horizontal, vertical) & volume of gel/buffer.
How can we measure the heat produced by the electrophoresis ?
Measure of produced heat: Power (W) = I * V = I^2 * R also known as Joule’s law
What happens to water during gel electrophoresis ?
What happen at the cathode/anode ?
It is electrolyzed.
Anode (+): oxygen and protons -> acidic
Cathode (-): Hydrogen and hydroxyl ions -> basic
What are the 2 functions of the buffer in electrophoresis ?
Buffer –> 2 functions:
- transport charge
- keep molecules uniformly charged throughout separation process
What are the compositions of TAE and TBE ?
TAE - 40 mM Tris-acetate, 1 mM EDTA
TBE - 89 mM Tris-borate, 2 mM EDTA
What is electroendosmosis (EEO) ?
EEO is the movement of liquid in a porous material due to an applied electric field.
How is agarose charged ?
What effect does this have ?
What can we therefore say about the net water flow ?
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.
What effect does high EEO have on migration speed ?
When EEO is high migration of DNA is retarded.
When should TAE buffer be used ?
- when DNA is to be recovered
- for electrophoresis of large (>10 kb) DNA
- low buffer capacity – recirculation
When should TBE buffer be used ?
- for electrophoresis of small (<1 kb) DNA
- increased resolution of small (1.5 kb) DNA
- high buffer capacity – decreased DNA mobility
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 ?
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
