Workshop II Flashcards
What are Isoschizomers?
restriction enzymes with the same recognition sequence, that originate from different species.
What are neoschizomers?
restriction enzymes that recognize the same sequence but generate different ends
What are isocaudomers?
restriction enzymes that recognize different sequences and yet generate the same ends.
What is the statistical frequency of a n-bp-long recognition site in the random sequence?
1/4^n bp
What is star activity and what factors promote it?
Star activity occurs when a restriction enzyme starts recognizing and cutting at recognition sites that closely resemble its recognition sequence but do not match it. Factors: - increased glycerol conc - suboptimal pH - suboptimal ionic strength - suboptimal divalent cofactor conc - presence of organic solvents( ethanol, DMSO) - prolonged reaction time - the amount of enzyme
How does DNA methylation influence the efficiency of enzymatic digest?
Some bacterial strains might be dcm+ or dam+, which means they are methylated on particular sequences. If these sequences overlap with the endonuclease recognition sequence than the efficiency of digestion is decreased.
What is the name and mechanism of reaction used to prepare the agarose gel?
Gelation is a reaction of adding water to the agarobiose powder. The agarobiose molecules fold into helixes, which form clusters that crate the porous structure of the gel.
How can fragment separation be regulated in electrophoresis?
The fragment separation is influenced by the avg pore size in the agarose gel, which can be regulated through the concentration of agarose in the solution.
What infuences the resistance of the electrophoretic solution and what is the result of the resistance?
Type of buffer and matrix, configuration (horizontal/vertical) and volume of gel&buffer solution. Resistance generates heat.
What happens to the different charged molecules during electrophoresis and how does it influence the pH distribution? What is the role of electrophoresis buffer?
Oxygen and H+ are made on anode (+) –> acidic pH
Hydrogen and OH- are made on cathode (-) –> basic pH
The buffer keeps the solution equally charged and with uniform pH.
What are the 2 buffer types and what are their features?
TAE:
- used when DNA is to be recovered
- better for separation of larger fragments
- low buffer capacity - need for recirculation
TBE:
- used for separation of smaller fragments (better resolution)
- high buffer capacity - decreases DNA mobility
What is electroendoosmosis?
The flow of the solvent molecules in the electri field.
The hydrated cations in the solution move in the opposite direction to DNA retarding the DNA migration.
What determines the voltage of electrophoresis?
Voltage is dependent on the length of the gel
What are the results of DNA overloading on the gel?
Smearing and trailing of the results.
What is the purpose of the gel loading buffer?
- increase density and add colour -> facilitate loading
- adding mobility dye -> aids tracking the progress of electrophoresis
