What type of matrix can be used for Ion Exchange Chromatography? -
ANSWER Insoluble matter (beads) containing covalently bound charged
residues
Anion exchange chromatography - ANSWER Negative proteins stick to
positive beads, only positive proteins go through
Cation Exchange Chromatography - ANSWER Positive proteins stick to
negative beads, only negative proteins go through
How do we choose a matrix based on the properties of the protein to be
isolated? - ANSWER Based on protein net charge, pI, pH and accessibility of
charged residues
How do we remove contaminants from the column? - ANSWER Wash buffer
How do we remove the protein from te column? (two ways) - ANSWER 1)
Increasing ionic strength of the buffer
2) Changing pH of the buffer
Variables in crystallization - ANSWER 1) Amount of starting material
2) Level of purity of the protein
3) Solvent (pH and concentration)
4) Precipitant (type and concentration)
Why is crystallization desirable? - ANSWER Allows to perform X-ray
crystallography
, How to test if crystals are proteins? (4 ways) - ANSWER 1) Dehydration
(dried salt crystal remains intact but protein one does not)
2) Crystals tend to be birefringent
3) Dye absorption (salt crystals don't absorb dye)
4) Run SDS-PAGE
Solutions used in lysis protocol - ANSWER Lysis solution, Neutralization
solution, Wash solution, and Elution Buffer
Lysis solution (alkaline plasmid isolation) - ANSWER SDS+Strong base
Disrupt cells' membranes
Neutralization solution (plasmid isolation) - ANSWER Precipitates (denatures)
genomic DNA
Wash Solution (plasmid isolation) - ANSWER Removes salt residues on
membrane
Elution Buffer (plasmid isolation) - ANSWER Dissolves DNA in slightly
basic buffer
How is plasmid DNA separated from genomic DNA? - ANSWER Denatured
(after lysis solution) genomic DNA can't reanneal as fast as plasmid DNA, thus
it gets precipitated.
Common features of usefuk plasmids (3) - ANSWER 1) Origin of replication
2) A drug-resistance gene
3) A region where DNA can be inserted without interfering with plasmid
replication or expression of the drug-resistant gene.
How is concentration of purified plasmid determined? - ANSWER UV Spec
(A260/A280 ratio should be 1.7-2.0)
How is number of plasmids determined? - ANSWER qPCR (a genomic gene
vs a copied gene)
How does uncut plasmid look on a gel? - ANSWER Two bands: One 'smaller'
than expected and one about as 'large' as expected
ANSWER Insoluble matter (beads) containing covalently bound charged
residues
Anion exchange chromatography - ANSWER Negative proteins stick to
positive beads, only positive proteins go through
Cation Exchange Chromatography - ANSWER Positive proteins stick to
negative beads, only negative proteins go through
How do we choose a matrix based on the properties of the protein to be
isolated? - ANSWER Based on protein net charge, pI, pH and accessibility of
charged residues
How do we remove contaminants from the column? - ANSWER Wash buffer
How do we remove the protein from te column? (two ways) - ANSWER 1)
Increasing ionic strength of the buffer
2) Changing pH of the buffer
Variables in crystallization - ANSWER 1) Amount of starting material
2) Level of purity of the protein
3) Solvent (pH and concentration)
4) Precipitant (type and concentration)
Why is crystallization desirable? - ANSWER Allows to perform X-ray
crystallography
, How to test if crystals are proteins? (4 ways) - ANSWER 1) Dehydration
(dried salt crystal remains intact but protein one does not)
2) Crystals tend to be birefringent
3) Dye absorption (salt crystals don't absorb dye)
4) Run SDS-PAGE
Solutions used in lysis protocol - ANSWER Lysis solution, Neutralization
solution, Wash solution, and Elution Buffer
Lysis solution (alkaline plasmid isolation) - ANSWER SDS+Strong base
Disrupt cells' membranes
Neutralization solution (plasmid isolation) - ANSWER Precipitates (denatures)
genomic DNA
Wash Solution (plasmid isolation) - ANSWER Removes salt residues on
membrane
Elution Buffer (plasmid isolation) - ANSWER Dissolves DNA in slightly
basic buffer
How is plasmid DNA separated from genomic DNA? - ANSWER Denatured
(after lysis solution) genomic DNA can't reanneal as fast as plasmid DNA, thus
it gets precipitated.
Common features of usefuk plasmids (3) - ANSWER 1) Origin of replication
2) A drug-resistance gene
3) A region where DNA can be inserted without interfering with plasmid
replication or expression of the drug-resistant gene.
How is concentration of purified plasmid determined? - ANSWER UV Spec
(A260/A280 ratio should be 1.7-2.0)
How is number of plasmids determined? - ANSWER qPCR (a genomic gene
vs a copied gene)
How does uncut plasmid look on a gel? - ANSWER Two bands: One 'smaller'
than expected and one about as 'large' as expected
