Molecular Techniques

    Cards (131)

    • DNA melting temperature (Tm) is defined as the temperature at which 50% of DNA strands dissociate.
    • DNA isolation involves the separation of DNA from cellular components such as organelles, lipids, proteins, carbohydrates, and DNA.
    • Different sources of DNA used for isolation include genomic DNA, single stranded DNA, vector DNA, and RNA.
    • DNA isolation can be done using various methods such as column chromatography, magnetic beads, and silica gel.
    • RNA isolation involves the separation of RNA from cellular components such as organelles, lipids, proteins, carbohydrates, and DNA.
    • Protein isolation involves the separation of proteins from cellular components such as organelles, lipids, proteins, carbohydrates, and DNA.
    • Sonication, which involves high intensity sound waves, is used in protein isolation.
    • Dialysis and ultrafiltration are procedures that separate proteins from small solutes.
    • Cells must be lysed before proteins can be isolated.
    • A membrane enclosing the protein solution is semipermeable, allowing the exchange of water and small solutes (glucose, salts, etc pass through the membrane freely but proteins do not).
    • Ion-exchange chromatography is a method based on electric charge.
    • Liquid Nitrogen is used in protein isolation.
    • Isoelectric precipitation is a method based on solubility of proteins.
    • Salting out is a method based on electric charge.
    • Cellular components include organelles, lipids, proteins, carbohydrates, and DNA.
    • Replication is a process in which the information in DNA is accurately replicated.
    • Transcription is a process where the DNA codes messenger RNA (mRNA).
    • RNA splicing is a process in eukaryotic cells where the mRNA is processed before migrating to the cytoplasm.
    • Tissues are homogenized in TRIZOL reagent, cells grown in monolayer are rinsed with ice cold PBS once, and cells grown in suspension are centrifuged to remove media and resuspended in ice cold PBS.
    • The DNA band is collected from the centrifuge tube, extracted with isopropanol, and DNA is precipitated with EtOH.
    • The aqueous phase is mixed with isopropyl alcohol to precipitate RNA, the pellet is washed once with 75% ethanol, and the RNA is dissolved in DEPC-treated water/TE buffer.
    • RNA (Ribonucleic acid) is a long single-stranded chain of phosphate and ribose units with nitrogen bases adenine, guanine, cytosine and uracil bonded to the ribose sugar.
    • Spectrophotometric analysis is used to determine the concentration and purity of RNA, with the A260/A280 ratio above 1.6.
    • DNA extraction involves lysing cells using a detergent, alcohol precipitating the lysate, resuspending DNA in CsCl and ethidium bromide, and centrifuging for hours.
    • The steps for preparation of RNA involve homogenization, phase separation, RNA precipitation, washing, and re-dissolving RNA.
    • Phenol, Chloroform and a chaotropic agent (guanidinium thiocyanate) are added to separate rRNA from ribosomal proteins.
    • Protein purification techniques are based on molecular size and include dialysis and ultrafiltration, density gradient centrifugation, and size-exclusion chromatography.
    • DNA extraction is time consuming, labor intensive, and expensive, making it inappropriate for routine use.
    • Translation is a process where mRNA carries coded information to ribosomes.
    • Proteins are involved in almost all biological activities, structural or enzymatic.
    • DNA is a double-stranded molecule consisting of a long chain of nucleotides, usually in a single-strand helix, with deoxyribose sugar, phosphate backbone, and adenine, guanine, cytosine, thymine bases.
    • RNA is a single-stranded molecule consisting of shorter chains of nucleotides, with ribose sugar, phosphate backbone, and adenine, guanine, cytosine, uracil bases.
    • DNA is fairly stable and can withstand alkaline conditions.
    • RNA is more reactive and is not stable under alkaline conditions.
    • This step is done multiple times to remove hemoglobin.
    • Urine is used as a non-invasive sampling approach for epidemiological research.
    • NaCl and CTAB solution (NaCl, 10% CTAB) added to supernatant and incubated at 70 ◦ C for 10 min (sample).
    • Samples are collected in the field and often no cold-chain is available, which can have a negative impact on the quality of the sample over time.
    • When thawed a standard red cell lysis buffer is added to the blood samples.
    • DNA has small grooves that provide protection from enzyme attachment.