Experimental methods Flashcards
Separation Method
Extraction
Extraction uses two layered fluids, one nonpolar and the other polar to dissolve a compound of interest.
If the compound of interest is polar it will dissolve in the polar layer whereas if nonpolar it will dissolve in the nonpolar layer. Like dissolves like.
Separation Method
Wash
Uses nonpolar and polar layers similar to extraction.
Dissolves impurities outside of the compound of interest, whereas in extraction the compound of interest is dissolved
Separation Method
Filtration
Isolate a solid from a liquid (like a Brita filter).
If interested in the solid, use vacuum filtration; if interested in liquid, use gravity filtration.
Separation Method
Crystallization
Used to purify an impure compound. Heat to high temperature, then after cooling at a slow rate, the pure substance will crystallize first
This occurs because impure substances have lower freezing points than pure substances
Generally will not result in a 100% pure compound.
Separation Method
Chromatography
Used to separate a mixture based on speed of movement through a medium.
The mixture is dissolved in a mobile phase fluid, usually liquid and nonpolar.
Then passes through a stationary phase structure, usually solid and polar.
Gel Electrophoresis
Gel
Agarose gel is used for larger molecules like nucleic acids
Polyacrylamide gel has smaller pores for proteins
Gel Electrophoresis
Southern blot
Used to detect a target sequence of DNA. A sample is run through gel electrophoresis before adding a fluorescent probe made of single-stranded DNA complementary to the target sequence.
Gel Electrophoresis
Northern blot
Similar to Southern blot but used to detect RNA.
Gel Electrophoresis
Western blot
Used to detect level of a target protein. Sample is separated through electrophoresis and then a fluorescent antibody specific to the protein is added.
The intensity of the electrophoresis band corresponds with the concentration of the target. A control protein, which has stable expression under different conditions, is used as a reference.
Distillation methods
Separates two liquids according to their boiling points. Heating too rapidly may cause poor separation.
Distillation methods
Distillate
Refers to the liquid with the lower boiling point.
Distillation methods
Simple distillation
Used for liquids with large differences in boiling point but generally will not result in a pure compound.
Distillation methods
Fractional distillation
Uses a column of glass beads to essentially cause repeated distillations. Compared to simple distillation, allows for purification of compounds with small differences in boiling points (<25oC)
Distillation methods
Vacuum distillation
Uses a low pressure environment in order to lower the boiling point of all liquids
This allows separation of liquids at lower temperatures, since some substances decompose at temperatures >150 oC
Chromatography methods
Paper
Chromatography methods
TLC
Chromatography methods
Gas
Chromatography methods
HPLC
Chromatography methods
Gel Filtration
Chromatography methods
Ion exchange
Chromatography methods
Affinity
cDNA cloning
DNA libraries
Two types of DNA libraries: genomic DNA libraries contain the whole transcript (including introns) while cDNA libraries contain specific genes, since they are made starting from mRNA.
Sanger Sequencing
Used to determine the sequence of DNA
Sanger Sequencing
Chain termination
Uses dideoxynucleotides (ddNTP), which are like nucleotides but missing 3’ -OH, to terminate chains.
In a large sample this results in different-length strands that terminate at all possible positions of the DNA.
Sanger Sequencing
Separation
Electrophoresis is then used to separate the strands based on length and determine sequence.
