Discussion 1 Flashcards
Polymerase Chain Reaction (PCR)
DNA
Purpose: Detecting and AMPLIFYING desired DNA fragments, creating mutations, and quantifying mRNA
Molecular Cloning
DNA
Purpose: Isolate and amplify a specific DNA fragment.
-Gene of interest amplified by PCR
-Usage of restriction enzymes to find and cut desired gene in plasmid
-Ligation
-Bacteria divide and amplify plasmid containing target gene
Quantitative RT-PCR (qRT-PCR)
mRNA
Purpose: Compared abundance of mRNA (gene expression) in different samples.
-Requires gene of interest AND a housekeeping gene for internal control
RNA-sequencing (RNA-seq)
mRNA
Purpose: Compares gene expression in multiple samples to show differences in expression between the samples
Fluorescent In situ hybridization (FISH)
DNA/RNA
Purpose: Detect specific sequences of nucleic acids (DNA/RNA) in tissues and cells using a fluorescent probe
Gel Electrophoresis
DNA/RNA
Purpose: A quick technique to separate DNA/RNA molecules by size
-Using an anode and a cathode on either end of a gel
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Protein
Purpose: Separates proteins by size using an upright gel
Western Blot
Protein
Purpose: After an SDS-PAGE, identify a single protein out of a mixture of thousands of proteins embedded in the PAGE gel (usually done with a housekeeping protein)
-Transfer: Transfer protein from PAGE to a nitrocellulose membrane via electropheretic current
-Detection: Incubate nitrocellulose membrane with antibodies against proteins of interest (either a tagged primary or a primary+tagged secondary)
Immunofluorescence (IF)
Protein
Purpose: Tagged antibody that is fluorescent
Immunoprecipitation (IP)
(Co-IP) Protein+protein, (ChIP) protein+DNA, (RIP) protein+RNA
Purpose: use antibodies to pull down specific proteins. If the pulled-down protein interacts with another protein, a second antibody can be used to demonstrate that interaction (co-immunoprecipitation)
-Then a western blot is typically done
Ion-exchange Chromatography
Protein
Purpose: positively charged proteins bind to negatively charged beads, negatively charged protein flows through
Chromatography
protein
Purpose: Separate and purify desired proteins out of a heterogenous cell lysate
Fluorescence Resonance Energy Transfer (FRET)
protein
Purpose: measure direct physical interaction or close proximity of two proteins
-use AFTER confirmation with other techniques
-Light emission from excitation is used to then excite another protein
-Orientation matters, working distance 1-10nm, choose the right fluorophores
Yeast Two Hybrid System
protein
Purpose: Evaluates direct physical interaction of two proteins, where the interaction forms a transcriptional activation complex (bait+prey)
-If the partners are compatable, then the prey will activate the transcription of a reporter gene
Gel-filtration Chromatography
protein
Purpose: Larger molecules go faster through large beads
Affinity Chromatography
protein
Purpose: Beads with attached substrates attract complementary enzymes which flow through the beads
Mass Spectroscopy
protein
Purpose: Highly sensitive technique to identify unknown proteins. Peptides are changed by an ionizer and accelerated. A detector detects proteins based on size and charge.
Flow Cytometry
protein
Purpose: Analyze physical and chemical characteristics of cells that pass through a single file tube and are detected by a laser.
Fluorescence-Activated Cell Sorting (FACS)
protein
Purpose: Sort a heterogenous mixture of cells into groups based on a flow cytometry characteristic like light scattering or flourescence.
-Desired cells are charged and sent through a tube where they are sorted into two different tubes based on having an either positive or negative charge
-Accurate, Fast, Robust, and Versatile.
