biol 355 lab techniques

fluorescence microscopy

increases contrast by labeling specific proteins or structure with fluorescent molecule. involves irradiating sample with light of specific excitation wavelength and capturing emitted light at a longer wavelength

fluorescent proteins ie GFP and RFP

GFP was originally isolated in a jellyfish, and is a genetically encoded tool to visualize gene expression patterns or subcellular protein localization. can be fused in fram to gene of interest to create fusion proteins. can monitor in living cells

FRAP- fluorescence recovery after photobleaching

technique used to monitor movement and kinetics of proteins in living cells. involves using intense laser beam to photobleach- irreversibly damage a fluorophore in small region, and observe rate at which unbleached molecules move into that are

2-photon microscopy

uses long wavelength light to simultaneously excite a fluorophore with 2 or more photons . offers advantage because it can penetrate more deeply and cause less damage and prevent out of focus light

electron microscopy

achieves 1000x better resolution than light microscopy, uses a beam of electrons which have shorter wavelengths than visible light. usually stain sample with heavy metal to provide contrast

cryo electron microscopy

samples are flash frozen in water (liquid ethane) to preserve native state before imaged with electron beam. multiple 2D images are reconstructed into 3D structure ie with microtubules

expansion microscopy

physically enlarges specimen by cross linking stained antibodies to matrix and expanding matrix in water. allows researchers to resolve details smaller than diffraction limit with light

blotting techniques

Extract, denature, electrophoresis, transfer to membrane, probe, detect, and separate molecules by size

northern blot

monitor RNA levels using labeled nucleic acid probes

southern plot

detect specific DNA targets

western blot

analyze proteins- samples treated with SDS and run on SDS page before being detected by antibodies

antibody staining- immunohistochemistry

primary antibodies to recognize specific epitope on target antigen, secondary antibodies coupled with fluorescent dyes bind to constant region of primary antibody to amplify signal

transcriptional reporters

placing reporter gene ie GFP under control of enhancer/ promoters from gene of interest. report where and when the regulatory elements are active- indicating gene expression patterns

DNA hybridization

relies on sequence specific base pairing between labeled probe and complementary target nucleic acid. basis of northern/ southern blotting and in situ hybridization

pulse chase- w/ radioactive amino acids

method to track transport of newly synthesized materials. cells are briefly pulsed with radiolabeled amino acids then chased with unlabeled ones, allowing researchers to monitor movement of labeled proteins through the cell over time

RNAi

knockdown technique that uses double stranded RNA to silence specific genes. Dicer cleaves long dsRNA into siRNAs which are then used by RISC complex to find and degrade complementary target mRNA

CRISPR-cas9

adapted from bacterial immune system, uses guide RNA to recruit Cas9 nuclease to specific DNA sequence. Cas9 creates double stranded break, which can be used to generate gene knockouts via error prone repair or specific modifications with homologous recombination

mutagenesis

introducing mutations into genome to study gene function. researchers may perform genetic screen to identify genes necessary for specific processes ie unfolded protein response

temperature sensitive alleles

conditional loss of function mutation- protein functions normally at permissive temperature but becomes inactive or misfolds at restrictive temperature

cellular fractionation

method to separate different cellular compartments based on size and density using differential centrifugation. successive rounds of centrifugation at increasing speeds allows specific organelles to be pelleted (larger components come out first)

biochemical fractionation

isolate individual proteins or complexes from cell lysates, usually using column chromatography to separate them by chemical/ physical properties

transgene

engineered genetic element introduced into an organisms genome

plasmid

small circular DNA vector used to deliver transgenes into bacteria/ eukaryotic cells

transfection

mechanical/ chemical process of delivering foreign nucleic acids into a cell

epitope/ epitope tagging

epitope is specific part of protein recognized by antibody, researchers often add short, defined sequences called epitope flags to allow them to be visualized using commercially available antibodies