Lectures Flashcards
Propagation of Error (or Propagation of Uncertainty)
Defined as the effects on a function by a variable’s uncertainty.
It is a calculus-derived statistical calculation designed to combine uncertainties from multiple variables to provide an accurate measurement of uncertainty.
Decellularization def and principle
process of removal of cells from their matrix using detergents.
- The hydrophobic tail of a detergent interacts with the hydrophobic part of the cell membrane of plant; thus, rupturing of the membrane takes place.
- This results in the release of cellular components to the outer side by making the extracellular matrix remain.
components in plant scaffolds
cellulose, hemicellulose, lignin, pectin
major advantage of the decellularization process
it can give support to the cells for their adhesion and growth and act as a template for the cells
also it improves the nutrient supply to the cells from the environment.
What are detergents, principle and how are they classified
Detergents are amphipathic molecules that can be classified into three categories based on their charge: nonionic, ionic, and zwitterionic detergents.
The hydrophobic tail of a detergent interacts with the hydrophobic part of the cell membrane of plant; thus, rupturing of the membrane takes place. This results in the release of cellular components to the outer side by making the extracellular matrix remain.
What are non-ionic detergents
Nonionic Detergents
Non-ionic detergents do not have any charged groups in their structures, and they are typically composed of a hydrophilic head group and a hydrophobic tail group.
These detergents damage the interactions between DNA and protein, lipid and lipid, and lipid and protein
Ionic detergents
Ionic detergents contain groups in their structures that allow them to solubilize charged molecules such as proteins.
These detergents make the cell and the nuclear membranes completely soluble in them and proteins will be denatured (wash away the prots in the sln)
Zwitterionic detergents
Zwitterionic detergents have both positive and negative charges in their structures, which allow them to maintain a net neutral charge.
Since these detergents are zwitterionic, their net zero charges on hydrophilic groups guard the protein state in the decellularization process and show the nature of ionic and nonionic detergents.
Solubilize and break membrane but protein intact
How to remove detergent from the cells
To remove the detergent fragments completely from the formed extracellular matrix (ECM), we use some salts to decrease the cleansing activity of detergents, and subsequently, the detergent is removed from the ECM.
* A commonly used salt for this purpose is CaCl2.
Characterization Techniques for a Scaffold
Swelling ratio test (estimate amount of swelling ->
proper transportation of nutrients)
- lyophilization, samples are weighed dry then
with PBS sln for several days
Degradation
- (mechanical strength to the cells, degrade itself
after some time)
Porosity
- Methods to determine the porosity of a scaffold.
Liquid-displacement method - ethanol as the -
solvent to find the scaffold porosity. (measure
volume of ethanol b4 and after having soaked
scaffold in it
Hydrophilic scaffolds
- allow the cell to grow and proliferate on their surface.
- If the contact angle is less than 90°, then the substance is said to be hydrophilic, and if it is more than 90°, it is said to be hydrophobic
What plant structural components are useful for scaffolds
Cellulose is a polysaccharide w stable glucose monomer
lignin and pectin can modify the behaviour of the gel
What does an increase of concentration of the detergent result in during the decellularization process
an increase in residue left in the matrix after the decellularization
Explain electrophoresis
When an electric field is applied across a gel or other matrix, charged molecules will migrate towards the electrode with the opposite charge. Smaller molecules typically move through the matrix more easily and thus travel further than larger molecules in a given time. (for example DNA neg charge)
Usually, a dye of high mobility is added; its migration serves to mark the progress of the experiment. You can also dye molecules to visualize them.
why might a dye of high mobility be added to electrophoresis gel
Usually, a dye of high mobility is added; its migration serves to mark the progress of the experiment.
dye also serves as a convenient measure of mobility (proportion of mobility of a component vs of the dye)
Molecules can also be dyed for visualization
What can electrophoresis be used for
Electrical charge differences can be used to separate and analyze mixtures of biopolymers
What is steady state for electrophoresis
When a charged particle is placed in an electric field, it is accelerated and its speed increases until its resistance becomes equal to the electric force. When the electric force and the resistance become equal, the speed of the particle becomes constant, which is often called steady state. In this case, electric force and resistance force are equal.
Supporting matrix electrophoresis
The kind of supporting matrix used depends on the type of molecules to be separated and on the desired basis for separation: charge, molecular weight, or both.
Almost all electrophoresis of biological macromolecules is at present carried out on either polyacrylamide or agarose gels. (poly cross-linked, agarose for large proteins)
PCR steps
(1) denaturation, in which double-stranded DNA templates are heated to separate the strands;
(2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and
(3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA.
what is pcr
PCR is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time.
what is melting temperature in pcr
The temperature at which half of the dsDNA is single-stranded is known as the melting temperature, Tm.
- During the denaturation step, the dsDNA melts opening up to ssDNA, and all enzymatic reactions stop (i.e. the extension from a previous cycle).
- For DNA denaturation, the temperature is usually raised to 93–96°C, breaking the H-bonds and thus increasing the number of non-paired bases.
The type of solvent, the salt concentration, the pH
the concentration of G/C and T/A can also affect the Tm value influence the denaturation process.
(G/C-rich 3 bonds higher Tm values)
Primers
The oligonucleotides used as primers typically consist of relatively short sequences (15–25 nt) complementary to recognition sites, flanking the segment of target DNA to be amplified
DNA polymerase
The extension phase is carried out across the target sequence by using a heat- stable DNA polymerase in the presence of dNTPs and MgCl2, resulting in a duplication of the starting target material.
- This enzyme has 5′ → 3′ DNA polymerase activity, i.e. it adds dNTPs from 5′ to 3′, reading the template from 3′ to 5′.
- For the majority of PCR experiments 1 min is sufficient to get a complete extension
Chemicals necessary in PCR
- Buffer-Stabilizes the DNA polymerase, DNA, and nucleotides
- DNA template-Contains region to be amplified,
- Primers-Specific for ends of amplified region, Forward and Reverse
- Nucleotides-Added to the growing chain
- Mg++ ions - Essential co-factor of DNA polymerase,
- DNAPolymerase-The enzyme that does the extension,TAQ or similar,Heat-stable,
Uses of PCR
Archaeology,
Diagnostic
Forensics
Wildlife conservation
GMO food detection
