Lab Final

1. Prewet the pipette tip to avoid lower delivery volumes. 2. Work at temperature equilibrium to minimize variation of volume.

1. Do not perform experiments unsupervised. 2. Wear goggles. 3. Ensure clothes cover the entire body. 4. No open-toed shoes. 5. Tie back hair.

pH reflects the concentration of hydrogen ions on a logarithmic scale. Acids partially ionize and donate hydrogen, thus lowering the pH of the solution.

Keeping a clean workspace is important for preventing contamination and ensuring accurate experimental results.

The acceptance of hydrogen ions, affecting pH

The pKa value, with smaller pKa indicating stronger acids and larger pKa indicating stronger bases

Resist pH changes, maintaining stability

The pH change during acid-base reactions

The concentration of absorbing species

Light absorption

Various methods, such as the Bradford method

Column, ion-exchange, size-exclusion, affinity, and high-performance liquid chromatography

Molecular mass and sequence amino acids

Charged fragments

Peptide synthesis

Size, as demonstrated with hemoglobin and vitamin B12 in the lab

PCR is used to amplify DNA segments using DNA polymerase, primers, dNTPs, and Taq polymerase.

The Sanger Method uses ddNTPs to interrupt DNA synthesis and determine DNA sequences.

DNA fingerprinting uses short tandem repeats (STRs) for paternity testing and restriction endonucleases to cleave DNA. The process involves isolating DNA, cutting with enzymes, gel electrophoresis, and Southern blotting.

X-ray crystallography produces electron density maps from protein crystals.

NMR measures nuclear spin to capture protein structure dynamics.

Carbohydrate purification methods include fractional precipitation, ion-exchange, size exclusion, and affinity chromatography.

Carbohydrate analysis methods include traditional chemical and enzymatic approaches, mass spectrometry, and high-resolution NMR spectroscopy.

Solid-phase oligosaccharide synthesis yields defined oligosaccharides useful in exploring lectin-oligosaccharide interactions.

DNA cloning involves obtaining a DNA segment, using cloning vectors, joining DNA fragments, moving recombinant DNA to host organisms, and selecting host cells containing recombinant DNA.

Gel electrophoresis techniques are used to estimate the number of different proteins in a mixture, degree of purity, isoelectric point, and approximate molecular weight of proteins in a sample.

SDS electrophoresis uses a detergent to give all proteins a similar charge-to-mass ratio and separates proteins by molecular weight.

Two-dimensional electrophoresis combines isoelectric focusing and electrophoresis to determine the isoelectric point and separate proteins by molecular weight, providing a more sensitive technique for protein analysis.

The shared proteins between species E and B, C, and D indicate a closer evolutionary relationship, leading to the placement of species E on its own branch in the phylogenetic tree.

Western blotting involves transferring proteins from a gel to a membrane and using antibodies to detect specific proteins, allowing for the identification and quantification of target proteins.

The Western blotting process includes steps such as adding primary and secondary antibodies, using enzyme-linked secondary antibodies, and adding colorimetric substrate to visualize the protein bands.

Troubleshooting steps for Western blotting include addressing issues such as extra bands, vertical lines, halo bands, absence of bands, presence of only prestained standards, bands with white circles or holes, smiling or frowning bands, and pale bands.

Melting curves can be used to estimate the C-G and T-A ratio of DNA by solving for the denaturation temperature ($T_m$), which provides insights into the stability and composition of DNA.

The lab involved the extraction and denaturation of proteins from muscle tissue, followed by gel electrophoresis to separate the proteins by molecular weight.

The lab included the use of Western blotting to identify myosin light chain proteins in the fish muscle extract.

Troubleshooting steps were applied during the lab to address issues related to Western blotting, such as the appearance of extra bands and pale bands.

Melting curves were used in the lab to estimate the C-G and T-A ratio of DNA by solving for the denaturation temperature ($T_m$), providing insights into the composition and stability of DNA.

The gene encoding the protein of interest is fused with a gene for an epitope tag. The protein is then precipitated by complexing it with an antibody that binds to the epitope. This immunoprecipitation helps identify associated proteins. The precipitated proteins can then be separated using affinity chromatography, and the proteins eluted from the column are identified by mass spectrometry.

The CRISPR/Cas complex consists of guided RNAs, trans-activating CRISPR RNA (tracrRNA), and one or more Cas proteins. The complex binds and destroys invading bacteriophage DNA through the action of the Cas protein nuclease.

CRISPR sequences consist of regularly spaced short repeats in bacterial genomes, while Cas proteins are nucleases or components of the immune system that have evolved to allow bacteria to survive infection by bacteriophages.

Current CRISPR technology requires one Cas protein (Cas9) to cleave DNA and a single guide RNA (sgRNA) to pair with the target DNA sequence and activate the nuclease domains. Cas9 has two separate nuclease domains, and the sgRNA is made of gRNA and tracrRNA fused into one RNA.

Mutation can be introduced by recombination when a DNA fragment enters a cell with CRISPR/Cas9 plasmids. CRISPR/Cas9 can be combined with other approaches such as RNA-Seq for additional information in research, medicine, genetic screening, and more.

Lipids are generally separated by differences in polarity or solubility in nonpolar solvents. The methods include adsorption chromatography using polar materials like silica, and gas chromatography to resolve mixtures of volatile lipid derivatives.

Neutral lipids are extracted with ethyl ether, chloroform, or benzene, while membrane lipids are extracted by ethanol or methanol. A commonly used extractant for membrane lipids is a mixture of chloroform, methanol, and water (1:2:.08).

Mass spectrometry allows the analysis of crude mixtures of lipids without prefractionation. It can determine the length of a hydrocarbon chain or positions of double bonds but not cis & trans.

Specific hydrolysis involves cleaving ester-linked fatty acids with mild acid or alkaline treatment. It can also cleave amide-bound fatty acids from sphingolipids under harsher hydrolysis conditions. Phospholipases specific for one of the bonds in a phospholipid generate simpler compounds.

Guided RNAs and tracrRNA play a role in pairing with the target DNA sequence and activating the nuclease domains of the Cas proteins in the CRISPR/Cas complex.

The Cas protein nuclease within the CRISPR/Cas complex binds and destroys invading bacteriophage DNA by cleaving it, thereby providing immunity to the bacterial host.

Mass spectrometry in lipid analysis allows the determination of the length of a hydrocarbon chain and the positions of double bonds, providing valuable structural information about lipids.

To prevent lower delivery volumes

To minimize variation of volume

Concentration of hydrogen ions

Pull the tip straight out

The strength of the acid or base

Concentration of absorbing species

Light absorption

Measuring protein concentration

Separating molecules based on size

Molecular mass

Sorting and measuring charged fragments

Peptide synthesis

pH change during acid-base reactions

To maintain stability of pH

Size-exclusion and affinity

Charged fragments

To determine the RNAs transcribed from a genome under specific conditions

To identify associated proteins

Cas protein and single guide RNA (sgRNA)

To determine the length of a hydrocarbon chain

It provides heat stability to the DNA polymerase

To cleave DNA at specific sites

With ethyl ether, chloroform, or benzene

Sanger Method

To destroy invading bacteriophage DNA

To separate DNA fragments by size

To visualize endogenous proteins

To produce electron density maps from protein crystals

By turning lipids into gas for elution

To capture protein structure dynamics

To pair with the target DNA sequence

Simpler compounds

Solid-phase oligosaccharide synthesis

To isolate specific DNA segments

Determining deleting or altering a protein to determine its function

To separate the precipitated proteins

To separate molecules based on size

To analyze carbohydrate structures

To amplify DNA segments

For paternity testing