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Explain how and why we sterilize equipment and solutions/buffers
Autoclave steam sterilizes equipment and solutions. standard process is 15-20 min at 121c.
explain the key steps in DNA extraction
DNA extraction involves cell lysis, removal of proteins and contaminants , Recovery of DNA and storage of DNA.
Give 2 examples of Plasmid purification methods
alkaline lysis, boiling prep method
Explain the principles of DNA quantification and appropriate use of equipment
DNA quantification involves measuring the concentration and purity of DNA using spectrophotometry. Proper equipment use includes selecting the right wavelength and ensuring accurate calibration for reliable results.
Define cell lysis give 3 methods
The process of breaking down the cell membrane and remove of cell wall to release cellular contents, including DNA. Methods include physical disruption, chemical lysis, and enzymatic lysis.
Give examples of ways to remove proteins and contaminants (4)
Salting out, Organic solvents (PCI), Ion exchange, Silica-gel membrane
how to Recover DNA
Precipitate genomic DNA with isopropanol, remove RNA using RNAse and wash the DNA pellet with ethanol to purify it.
Define Storage of DNA and what temperature
using TE-Buffer or MilliQ and stored as a pellet at -20c
explain alkaline lysis
The NaOH increases the pH to 12 and denatures the Genomic DNA irreversibly. Plasmid DNA is reversibly denatured and later renatured.
explain boiling mini prep
Heating causes breakage of cell wall, denatures of DNA and proteins, plasmid DNA is reversibly denatured and genomic DNA is irreversibly denatured.
Explain how PCR works and Design a PCR
PCR is a technique used to amplify specific DNA sequences through repeated cycles of denaturation, annealing, and extension. Designing a PCR involves selecting appropriate primers, determining the optimal annealing temperature, and defining the number of cycles for amplification.
Name and explain the importance of the different components of a PCR
Template, Primers, DNA polymerase, Buffer dNTPs Mg.
Explain the phases, steps, and cycling in a PCR
Initial, exponential, log linear, plateau.
Name 8 different types of PCR
End point, Real time (qPCR), Degenerate, Nested, Reverse transcription, colony, Touchdown, Gradient.
what are plasmids and how are they used in the lab
Double stranded circular extra chromosomal DNAmolecules that replicate independently of chromosomal DNA. They are commonly used as vectors for cloning, gene expression.
what are the key features on a lab plasmid
Key features of a lab plasmid include an origin of replication, selectable markers, multiple cloning sites (MCS), and promoter regions for gene expression.
how does blue/white selection work
Blue do not have clone, white do
Name the major molecular biology enzymes (7) and describe their use in the lab
Type II restriction endonuclease (cleave DNA at specific Nucleotide sequence), PCR enzymes, DNA ligase (join 2 DNA fragments), Reverse transcriptase (synthesize complementary DNA using RNA template), DNase I (Removes DNA from a RNA Preparation), RNase A (removes RNA from a DNA preparation), RNase H (removes RNA from RNA/DNA hybrids).
explain how restriction endonuclease are used
Restriction endonucleases are enzymes that cut DNA at specific nucleotide sequences.
How is agarose gel electrophoresis used to study and analyze DNA
Can you explain what you see on an agarose Gel
how is a ligation performed
Joining linear fragments of DNA together with the formation of covalent bonds.
how is bacterial transformation performed with plasmid DNA
Chemical or physical methods are used to introduce plasmid DNA into bacterial cells, allowing for the uptake and expression of foreign genes.
Explain how traditional cloning and PCR cloning work
Traditional cloning involves the insertion of a DNA fragment into a vector, followed by transformation into a host organism for replication. PCR cloning amplifies the desired DNA fragment using polymerase chain reaction, then ligates it into a vector for transformation.
what is sub cloning
moves DNA fragments from one plasmid to another plasmid.
Name the different first generation sequencing methods (5)
Plus minus method (Sanger and Coulson), maxim Gilbert, chain termination method (Sanger), fluorescent dye-based Sanger sequencing, commercial automated sequencing machines.
Describe Sanger and automated Sanger sequencing and how they work.
Sanger sequencing, developed by Frederick Sanger, uses chain-terminating nucleotides to create DNA fragments of varying lengths, which are then separated by capillary electrophoresis to determine the sequence. Automated Sanger sequencing utilizes fluorescent dyes attached to the nucleotides, allowing for real-time detection and analysis of the DNA fragments in a single run.
Explain why we purify RNA
RNA is extremely sensitive to ribonuclease degradation and must be purified to ensure integrity for downstream applications such as reverse transcription and quantitative PCR.
What precautions are taken to ensure good quality RNA (4)
Always wear gloves, treat all solutions with DEPC, autoclave instruments, and buffers with tris twice, use RNAse away.
Explain how RNA is purified
RNA purification involves isolating RNA from cellular components using methods like phenol-chloroform extraction or column-based approaches. These techniques remove proteins, DNA, and other contaminants to obtain high-quality RNA suitable for downstream applications.
Explain RNA gel electrophoresis
RNA gel electrophoresis is a technique used to separate RNA molecules based on their size by applying an electric field to a gel matrix, allowing visualization and analysis of RNA integrity and quantity.
Can you analyze and interpret an RNA gel
The larger rRNA band should be approx. twice as intense than the lower rRNA band, Degraded RNA will be a smear with a very intense smear at the bottom.
Describe their general design of next generation sequencing
DNA fragmentation, DNA adapters ligated to end pieces of DNA, fragments immobilized on a solid surface.
name (2) and explain 2nd generation next generation sequencing platforms
Pyrosequencing (fragments DNA into 400-700bp fragments, ligate DNA adapters, Anneal to beads, then amplification), illumina (fragment DNA, ligate DNA to adapters, cluster generation using bridge amplifcation, sequence using fluorescent tagged nucleotides).
name the 3rd generation next generation sequencing platforms (2) and describe the basic mechanisms.
Minion (dsDNA unwound into ssDNA, as ssDNA passes through the ion channel current changes, each nucleotide affects the current flow rate differently), Single Molecule Real Time sequencing (SMRT) (DNA not fragmented or amplified, Hairpin loop ligated to linear double stranded DNA template, Denatured to produce a circular single stranded DNA,which is sequenced in real time as polymerase incorporates nucleotides.)
Explain how qPCR works (5 steps)
isolate RNA, Design gene specific primers, RT PCR with generic primers or gene specific primers, Run qPCR, analyze
Explain the key aspects in primer design for PCR in general but also qPCR specifically
Key aspects in primer design include selecting appropriate primer length (usually 18-25 nucleotides), ensuring a melting temperature (Tm) that is optimal for the reaction (typically 55-65°C), avoiding secondary structures like hairpins, and ensuring specificity to the target sequence while minimizing the chance of non-specific binding.
Understand and explain qPCR methods of quantification
Quantification methods in qPCR include absolute quantification using standard curves and relative quantification comparing target gene expression to a reference gene.
explain how different dyes and probes are used in pPCR
DNA binding dyes (binds to dsDNA), Hybridization probes: Fret (3 or 4 primer approach, with fluorophores and quenchers. -molecular beacons (2 primers and 1 gene specific probe, quencher, and reporter at each end of probe, R and Q separate when annealed increasing fluorescence). -Scorpion probes (a single primer with a gene specific probe that forms a loop, releasing fluorescence upon hybridization)
in broad terms explain sage and microarrays
SAGE is only able to monitor gene expression of one gene or very few genes with northern blotting or reverse transcriptase PCR, can determine expression of many genes, but only genes being expressed. quantitative and qualitative. labour intensive. Microarrays allow for the simultaneous analysis of thousands of genes by hybridizing cDNA to a grid of oligonucleotide probes. This technique enables the measurement of gene expression levels across a genome, providing a comprehensive overview of cellular activity.
explain the differences between the major types of arrays (6)
spotted arrays are less expensive
spotted arrays are customizable
spotted arrays have more variability between arrays
genechips have standardized protocols
spotted arrays use individual probe (gene) to calibrate, whereas genechips use a larger number of probes to calibrate
genechips have a greater range of magnitude to detect transcripts.
can you explain in general how gene expression is monitored
Gene expression is monitored through various techniques such as quantitative PCR, microarrays, and RNA sequencing. These methods assess the transcription levels of genes, allowing researchers to understand gene activity and regulation within cells.
Explain the general concept of ddPCR
ddPCR, or digital droplet PCR, is a highly sensitive technique that enables precise quantification of nucleic acids by partitioning a sample into thousands of droplets, allowing for the detection of rare mutations and low-abundance targets with high accuracy.
Explain in general how ddPCR analysis is performed
Droplet digital PCR (ddPCR) analysis is performed by partitioning a sample into thousands of droplets, each containing a limited number of target DNA molecules. This allows for precise quantification of DNA by counting positive and negative droplets after amplification, providing a highly sensitive and accurate measurement of gene expression.
How is ddPCR applied (6)
Detecting low copy number DNA, genotyping single cells, Absolute quantification, gene expression, NGS library preparation, genetic linkage studies.
how is NGS used to understand human disease
Next-generation sequencing (NGS) is used to identify genetic variants associated with diseases, enabling researchers to understand the genetic basis of conditions, discover biomarkers for diagnosis and treatment, and explore the role of mutations in disease progression.
generally explain the concept of GWAS
linking a genotype to a disease by comparing populations with a tendency to develop a disease to a population that does not.
