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Lab Tech

First Aid - Biochem - Lab Tech

PCR - basics amplifies a desired fragment of DNA, 1.denaturation, 2.annealing, 3.elongation > these steps repeated multiple times for DNA sequence amplification *agarose gel electrophoresis = size separation of PCR products (smaller molecules travel further)
PCR - 1.denaturation DNA is denatured by heating to generate 2 separate strands
PCR - 2.annealing during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified
PCR - 3.elongation heat-stable DNA polymerase replicates the DNA sequence following each primer
Blotting procedures - SNoW DRoP Southern = DNA Northern = RNA Western = protein
Southern blot DNA sample electrophoresed on a gel and then transferred to a filter - soaked in denaturant and exposed to labeled DNA probe that recognizes and anneals to complementary strand - double stranded labeled piece of DNA is visualized filter is expo to film
Northern blot similar to Southern except that Northern blotting involves radioactive DNA probe binding to sample RNA
Western blot sample protein is separated via gel electrophoresis and transferred to a filter, labeled antibody is used to bind to relevant protein
Microarrays thousands of nucleic acid sequences are arranged in grids on glass or silicon - DNA or RNA probes hybridized to chip, scanner detects relative amounts of complementary binding, used to profile gene expression levels or to detect SNP
ELISA (enzyme-linked immunosorbent assay) -rapid immunologic tests for antigen-antibody reactivity - often used for HIV testing to find anti-HIV, sensitivity and specificity of ELISA approach 100% but both false positives and negatives do occur
ELISA method 1.test antigen (coupled to color generating enzyme CGE)to see if recognized by immune sys. or 2.test antibody (with CGE)to see if a certain antigen is present - if target is present test solution will have an intense color reaction = positive test result
FISH (fluorescence in situ hybridation) fluorescent DNA or RNA probe binds specific gene at site of interest - used for specific localization of genes, direct visualization of anomalies(microdeletions) at molecular level (when deletions are too small to be seen by karyotype) *fluorescene-gene +
Sanger DNA sequencing dideoxynucleotides halt DNA polymerization at each base, creating sequences of various lengths that encompass entire original sequence - terminated fragments are electrophoresed and the original sequence can be deduced
Karyotyping metaphase chromosomes are stained, ordered, and numbered based on shape, size, banding, performed on blood, bone marrow, aminotic fluid, placenta - dx. chromosome imbalances (trisomies, deletions, sex chromosome disorders)
Created by: Smukadam



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