Four major classes of biological molecules that play essential roles in all organisms.
- Nucleotides
- Amino acids
- Carbohydrates
- Lipids
Model organism
A model organism is a non-human animal or plant species that is conveniently studied to understand particular biological phenomena , with the expectation that discoveries made in the model organism will provide insight into the working of an organism of interest.
Most commonly use model organisms
- Fruit fly ( Drosophila melanogaster)
- Hermaphrodite worm (Caenorhabditis elegans)
- Arabidopsis thaliana plant
- Mouse model (Mus musculus)
When do you use centrifugation and cell fractionation.
When you would like to isolate different organelles, or separating soluble and membrane-associated molecules from other cellular components.
What are the scientific principles of the centrifugation and cell fractionation.
- Intact cells must be disrupted to release their contents.
- Individual components can be separated by the centrifuge.
What methods can be used to release cell contents from a cell.
- Ultrasonic waves
- High pressure
- Detergents that generate holes in the cell membrane
How does the centrifuge work?
The centripedal forces that results when samples are accelerated at upwards of 100000g causes large molecules to move toward the bottom of the tube and form a pellet.
Differential centrifugation.
Repeated centrifugation at progressively higher speeds will fractionate cell homogenates into their components.
How Gel electropheresis works.
The negative charge of DNA and RNA molecules are used too seperate the DNA and RNA molecules of different sizes. This method relies on a porous gel made from agarose or polyacrylamide through which nucleic acids migrate when the gel slab is immersed in buffer and an electrical field applied.
How do we make sure proteins seperate in gel electrophoresis in a correct way?
The proteins must first be denatured and then coated with a negatively charged detergent.
What are Blotting techniques used for?
They are used by scientists to detect specific RNA,DNA or protein species in a sample.
What are subtypes of blotting techniques such as?
- Southern blotting
- Northern blotting
- Western blotting
- South-western blotting
- Eastern blotting
Why are seperated molecules transferred to a membrane after gel electrophoresis.
Proteins and nucleic acids have difficulty traveling through a gel. Once a protein or nucleic acid sample has been separated, you will need to readily access the separated molecules for detection with a nucleic acid probe or antibody. By transferring the molecules to a solid, sticky surface, they can easily be exposed to the probe or antibody you are using to detect a specific sequence or protein.
Sanger sequencing
- Production of chain-terminated DNA fragments from a common template, where each terminated chain incorporated a dideoxynucleotides (ddNTP) with a specific fluorescent label.
Dideoxynucleotides
Dideoxynucleotides or ddNTPs are nucleotides lacking a 3’-hydroxyl (OH) group on their deoxyribose sugar. This results in termination of elongation because the dideoxynucleotides cannot bind another dNTP.
What is gene cloning ?
Gene cloning involves taking a piece of DNA from the organism where it naturally occurs and putting it into a host cell such as the bacterium.
Why is it important to have selectable markers on cloning vectors.
Because only about one in a million of the bacterial cells successfully take up the plasmid.
When do you use recombinant protein expression.
You use it when you wish to have a physical sample of a pure protein of interest for an assay.
Steps of recombination protein expression.
- PCR products are inserted expression vectors.
- Recombinant vectors are inserted into E.coli.
- Recombinant E.coli are planted on antibiotic-enriched media and DNA is sequenced into a protein in the recombinant E.coli.
- Protein is extracted, it can be run on an SDS-PAGE gel to be visualized.
When do I use PCR.
- To detect the precence of a DNA sequence in a sample.
- Amplify and Isolate a gene for cloning
- Amplifying a gene so that it can be sequenced and studied.
- RT-qPCR is used when you want to quantify the expression of a gene at RNA level.
Southern blotting steps
- Nucleic acids seperated according to size by electrophoresis in an agrosegel.
- Separated nucleic acids blotted onto membrane by suction of buffer through both gel and paper.
- Membrane with seperated nucleic acids hybridized with labeled probe.
- After removal of unbound probe, bands complementary to labeled probe are revealed by specific detection of label.
What questions do nuclear run-on assay answer.
- Is a particular gene regulated primarily at the level of transcriptional initiation, or regulated by downstream events.
- How does the rate of transcriptional activation/ initiation compare under different experimental conditions, or compare between different genes.
Advantage over nuclear run on assay
- Resulting are not influenced by RNA degration, since degradation takes place in the cytoplasm, but radioactive labelling is preformed within the isolated nuclei.
- By removing cytoplasm, the large pool of unlabelled NPTs is removed, therefore more labelled.
When and why would i perform RT-qPCR.
When we want to compare levels of one or more transcripts between samples with high accuracy and high sensitivity. It can detect very low concentration of cDNA that northern Blots or DNA microarrays cannot.
