Modul 3 - Practicalcourse - WS2324

Modul 3 – practical training – WS 2023/24
Buffers
 The purpose of a buffer in a biological system is to maintain intracellular and extracellular pH

within a very narrow range and resist changes in pH in the presence of internal and external
influences (addition of acids or bases).
 constant pH is important for proper function of enzymes (example: blood pH 6,8 – 7.4),
bigger changes can permanently damage enzymes with adverse consequences
 Buffers consist of a pair of a weak acid and its conjugate base
 effective buffer: number of moles for weak acid and conjugated base have to be sufficiently
higher compared to the amount of added acid or base. Best buffering occurs at a 1:1 ratio
(mind the capacity of a buffer, known 1-2 important buffer systems in biology)
Cell culture
 = Maintenance and growth of cells in vitro in a lab

 primary cell culture  restricted life time; immortalized cells (e. g. by spontaneous mutation,
tumor, hTERT)  infinite proliferation
 suspension cells float around in the medium, adherent cells stick to surface and need to be
enzymatically or mechanically mobilized before handling (eg. by trypsin)
 cell culture medium with growth factors and nutrients, cultivation in a humified atmosphere
 disadvantages: Less physiological: reduced intercellular communication, missing extracellular
matrix, … ,  3D spheroids and organoids as new developments
Restriction enzymes
 enzymes (endonucleases) that cut DNA at specific sequence motifs (palindromic, motifs of
varying sizes, mostly 4 -8 basepairs, create different ends (blunt end, sticky))
 defense system of bacteria against foreign DNA
 used for DNA manipulation in molecular biology
 nomenclature according to the bacterium of origin (EcoRI from E. coli)
Plasmid
 small (up to around 10000 bp), circular, double stranded piece of DNA
 naturally occurring in bacteria and yeast
 autonomous replication, additional information
 in nature: Horizontal gene transfer (evolutionary benefits, e.g. adaption to environment)
Exchange by conjugation or cytoplasmic bridges
 In molecular biology/biotechnology: Production of RNAs or proteins of interest in bacteria,
plants and mammalian cells
 Main components: ori of replication, multiple cloning site, promotor, resistance gene
DNA isolation
 steps:
- disruption of cellular structures to create a lysate (physical: grinders, metallic or ceramic
beads to disrupt cells or tissues, or sonication to disrupt tissues and lyse cells, chemical:
detergents (e.g., SDS) and chaotropes (e.g., guanidine salts and alkaline solutions),
enzymatic: lysozyme, zymolase and lyticase, proteinase K, collagenase and lipase, among
others)
- precipitate proteins and separate the soluble DNA from cell debris and other insoluble
material
- binding the DNA of interest to a purification matrix (spin column), wash and elute or remove
residual proteins and further hydrophobic components via phenol/chloroform extraction
(often done for genomic DNA)
- precipitate (ethanol or isopropanol)
DNA analysis
 for size and integrity: agarose gel electrophoresis (agarose gel, running buffer, sample
loading buffer to track the DNA and to allow sinking in the gel, electricity, Smaller molecules
travel faster than larger molecules in gel), degradation visible as smear
 for amount and purity: absorbance (260 nm: DNA/RNA, 280 nm: proteins, 230 nm:
chaotropic salts), fluorescence (addition of a DNA binding fluorescence dye, more sensitive
than absorbance, but standard curves necessary)
 PCR (see below), sequencing
Polymerase chain reaction (PCR)
 PCR is a method used to make millions to billions of copies of a specific DNA section
 required components:
- a DNA template that contains the DNA target region to amplify
- a DNA polymerase (= an enzyme that adds nucleosides to a growing DNA strands according to
the template strand (heat-resistant Taq polymerase is common)
- two DNA primers that are complementary to the 3' ends of each of the DNA target strands
- deoxynucleoside triphosphates (dNTPs)
- a buffer solution providing a suitable chemical environment for optimum activity and stability
of the DNA polymerase and bivalent cations (Mg2+, Mn2+) for polymerase fidelity and efficiency
- procedure: denaturation (96 oC); annealling (50-60 oC), synthesis/primer elongation (72oC), 35-
40 cycles
 degenerated primers can be used to introduce DNA mutations
transformation
 Bacterial transformation is a natural process in which cells take up foreign DNA from the
environment at a low frequency
 With the advent of molecular cloning in the 1970s, the process of transformation was
exploited and enhanced to introduce recombinant plasmid DNA into bacterial strains that were
made “competent,” or more permeable, for DNA uptake. Transfection is a modern and
powerful method used to insert foreign nucleic acids into eukaryotic cells.
 most common methods:
- chemical competence: The procedure showed increased permeability of the bacterial cells to
DNA after treatment with calcium (Ca2+) and brief exposure to an elevated temperature,
known as heat shock.
- electroporation: an electrical field is applied to the cells to enhance the uptake of DNA (short
pulses of high-voltage electric fields). Such electric fields are believed to increase the cell’s
membrane potential, thereby inducing transient permeability to charged molecules like DNA.
- liposome-mediated transfection (also lipofection): uses a lipid complex to deliver DNA to
cells, the technology uses tiny vesicular structures called liposomes that have the same
composition as the cell membrane; DNA/RNA/proteins can be enclosed in such liposomes.
Depending on the liposome and cell type, the liposome can be endocytosed or directly fuse
with the cell membrane to release the DNA construct into cells
- viral transfection: introduction of viral DNA (previously manipulated) into plants and
mammalian cells (e.g. lentivirus) by virus infection
molecular cloning
 Molecular cloning is an essential technique to create DNA-based experimental tools for

expression in bacterial, plant or mammalian cells.
 workflow:
- Choice of host organism and cloning vector
- Preparation of vector DNA (plasmid isolation, restriction digest)
- Preparation of DNA to be cloned (amplification by PCR, excision from donor DNA)
- Creation of recombinant DNA with DNA ligase
- Introduction of recombinant DNA into host organism (transformation, transfection)
- Selection of organisms containing vector sequences (marker introduced by plasmid, eg.
antibiotics resistance, fluorescent protein)

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Modul 3 – practical training – WS 2023/24

 The purpose of a buffer in a biological system is to maintain intracellular and extracellular pH

 = Maintenance and growth of cells in vitro in a lab

Polymerase chain reaction (PCR)

 Molecular cloning is an essential technique to create DNA-based experimental tools for

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