BME 205 Lab Final

What are the 3 micropipette models and how much does each measure?

L20 (20μ), L200 (200μ), and L100 (1000μ)

How to use micropipette first and second stops?

Push down to the first stop, insert tip into the liquid, slowly release plunger until the tip is full and then remove from liquid, dispense the liquid by slowly pushing to first stop, pause, then continue to second stop

What is pH?

pH is a measure of a solution's acidity which reflects the amount of hydrogen ions are in the solution

Acid vs. base

Acids donate hydrogen ions while bases accept hydrogen. Acids have a pH < 7 and bases have a pH > 7, pH 7 is neutral

What does a pH meter measure?

The pH meter uses electrodes to monitor the voltage changes causes by different concentrations of hydrogen ions. This is done by measuring the potential between a pH sensing electrode and a reference electrode

Precision vs. accuracy

Precision is how close the measured values are to one another and accuracy is how close the measured values are to the true/accepted value

Standard deviation

A measure of how spread out the data is in relation to the mean

Standard deviation formula

Square root of the sum of the (data value minus the mean) squared over the \# of data points minus 1

Serial dilution

A technique of making an initial dilution and creating further dilutions with that diluted sample

What is the final diltuion of 10g of NaCL per mL with 1/10 being the first dilution, and then 1/4 serial dilution 4 times?

1/10 * 1/4 * 1/4 * 1/4 * 1/4 = 1/2560

DH5-alpha cells

Genetically engineered E.coli cells that are used from plasmid transformation and general closing applications

BL-21 cells

Protease deficient genetically engineered E.coli cells used for protein expression

Bacterial growth

The growth of inoculant bacteria in a lab setting that goes through four distinct phases

Lag phase

The time where bacteria take time to adjust to the environment they are placed in

Exponential phase

The time where bacteria are in a nutrient rich environment and the bacteria increase enzyme production and growth + divide at a constant rate

Stationary phase

The time were metabolic processes occur at a constant rate until it hits a plateau in growth

Death phase

The time were nutrients are depleted, toxins accumulate, cell viability decreases, and the population declines exponentially

Generation time

The time is takes for 2 new cells to arise from an original cell

Generation time formula

GT = (time interval)/(3.3log(population after t/initial population))

Steps for plasmid DNA isolation

First, re-suspend pelleted bacteria in buffer 1 and vortex to ensure resuspension, add 200μl of buffer 2 and invert until pink, clear, and viscous and then incubate for 1 minute, add 400μl buffer 3 to neutralize the lysate and invert until yellow and precipitate forms, centrifuge for 5 minutes, apply supernatant to spin column, centrifuge for 60 sec, and discard the flow thru, re-insert column in collection tube and add wash buffer 1, centrifuge for 60 sec and discard flow thru, add 400μl of wash buffer 2, centrifuge for 60 sec and discard flow thru, add 50μl double distilled water to the center of matrix, spin for 1 min to elute the DNA, finally place the tube on ice

Buffer 1 (plasmid resuspension buffer)

Buffer 1 contains Tris-HCL and EDTA to re-suspend the plasmid. The Tris is pH 8 and acts as a buffering agent. EDTA soaks up cations which destabilizes bacterial lipid membranes and inhibits DNAases

Buffer 2 (plasmid lysis buffer)

Buffer 2 contains NaOH and SDS to lyse the plasmid. NaOh helps break down the bacterial wall to let the DNA release from the cell and disrupt H bonds. SDS gives the solution a negative charge to break down the lipid membrane

Buffer 3 (plasmid neutralization buffer)

Contains guanidine hydrochloride and RNAase to neutralize the lysate. It aids in precipitation of proteins, chromosomal DNA, debris, and SDS. It also helps plasmid DNA reversibly bind to silica. The RNAase enzymatically degrades all RNA

Wash buffer 1

Contains guanidine hydrochloride isopropanol which helps secure plasmid DNA to the silica. The isopropanol dissolves salts and contaminants

Wash buffer 2

Contains Tris-HCl at pH 7 and it removes chaotropic salts without disrupting bonds between DNA and the silica membrane

Elution buffer with Tris/EDTA vs with water

With Tris/EDTA, DNA is stable for a longer time than water. Water is best if DNA is used in salt-sensitive applications

260/280 ratio

A ratio of ~1.8 is accepted as "pure" DNA. A lower ration may indicate that presence of contaminatns

260/230 ratio

A ratio of 2.0-22 is accepted as "pure" and is a secondary measure of DNA or RNA purity

TBE buffer

This buffer is made of Tris, boric acid, and EDTA for gel electrophoresis and other molecular biology applications

What are the 4 nitrogenous bases and base pairs?

A (adenine), T (thymine), G (guanine), and C (cytosine). The base pairs are purines (AT) and pyrimidines (GC)

Directionality

Arises from phosphate-deoxyribose backbone in which the 5' end of the nucleotide is connected to the 3' end of the preceding nucleotide through a phosphodiester bond allowing for base pairing

Base pairing

Pairing A with T and G with C to create DNA strands of the complement bases

DNA synthesis

Occurs at the replication fork and is initiated by helicase that unwinds the DNA by breaking the hydrogen bonds between the base pairs and forms complementary single stands of DNA. RNA polymerase synthesizes RNA complementary to each DNA strand at the replication site, creating a priming sit for DNA polymerase to attach and synthesize DNA by making the complementary base pairs. The RNA primers have 3' OH which attaches to 5' phosphate of the incoming nucleotide base

Where does DNA synthesis occur?

The the replication fork

What does helicase do?

It initiates synthesis by unwinding the DNA strands into two separate strands by breaking the hydrogen bonds between base pairs.

RNA polymersase

Synthesizes RNA complementary to each DNA strand at the replication site and creates a priming site for DNA polymerase

DNA polymerase

Attaches to priming site and synthesizes the DNA by making the complement base pair

RNA primer

Contain 3' OH to attach to the 5' phosphate of the incoming nucleotide base

What is PCR (polymerase chain reaction)?

It is a technique used to amplify and replicate a specific DNA sequence

What are the components of a PCR?

DNA sample to be replicated, primers, buffer to maintain pH 7.5, DNA Taq polymerase o replicate the DNA, magnesium

What are the 3 PCR cycle phases and required temperatures?

Denaturation at 95 degrees to separate the strands, annealing (3-5 degrees cooler than the primer melt point) to form hydrogen bonds with the primers, and replication at 70-72 degrees for dNTP to bind to the component

dNTPs (deoxynucleotide triphosphates)

Contain deoxyribose and provide single bases to go into the DNA, added to the 3' end of the primer

What are the PCR primers?

Forward and reverse primers that bind to their DNA sequence of interest and amplify GFP, the primer must have a specific length

Restriction enzymes

Proteins that cleave DNA into fragments at or near the specific recognition sites within DNA molecules, they are helpful for cutting DNA into sizes suitable for cloning

Blunt ends

When the enzyme cuts straight across the double helix

Sticky ends

When the enzyme cuts in an offset fashion

What temperature do restriction enzymes cut?

About 37 degrees

Agarose gel electrophoresis

A technique used to determine the length and purity of DNA/RNA and amino acid molecules

What are the components of agarose gel electrophoresis

Agarose as a separation medium, a dye to visually track migration, EDTA to bind to unwanted metal and inhibit metal and protect DNA, glycerol to ensure the DNA in the ladder and sample sinks below the buffer

Describe "run to red" and gel electrophoresis polarity

It describes how the electrical current moves the DNA in the positive direction through the gel, the DNA backbone leave a negative charge

How to monitor DNA in gel electrophoresis?

The DNA sample is mixed with tracking dyes to monitor the DNA and the gel will contain bands that shoes how many fragments are present and how large they are relative to eachother

Transformation

Process in genetic material is incorporated within a bacterial cell

What is the process of transformation?

Treat the bacterial cells with calcium chloride to prepare bacteria to become competent cells, the competent cells are mixed with the ligated DNA and heat shocked at 42 degrees to transform the plasmid into the competent cells

Where does the bacteria go after transformation?

It is plated on an agar plate with antibiotic and screen for colonies with the insert plasmid

Competent cells

A cell's ability to take up foreign DNA from its surrounding environment

Why is ampicillin used with the agar plate?

Ampicillin is an antibiotic used to selectively eliminate bacteria that have not been transformed with plasmids containing an ampicillin resistance gene

Why does the antibiotic used for plating matter?

The antibiotic used depends on if the plasmid contains the gene resistant to that antibiotic

Ligation

The joining of two DNA fragments together by enzymatic means

Ligases

Repair DNA that is broken/damaged

Why is the ligated gene important for transformation?

The ligated gene is inserted into the competent cells through transformation

Hind III and EcoRI

Restriction enzymes that are used to allow the insert plasmid to be put into the backbone properly

What are the components of pRSET plasmid?

His-tag, multiple cloning sites, T7 promoter, RBS, start codon

His-tag

Used to purify a tagged protein and binds to nickel

Cloning sites in pRSET

Contains a number of restriction enzyme sites to allow ready addition of an insert of interest

T7 promoter

A sequence of DNA that initiates transcription on the expression vector

RBS (ribosome binding site)

A sequence where the ribosome can bind during the initiation of translation

Start codon

The first codon of the transcribed mRNA translated by a ribosome

Sanger sequencing

A method in which chemical termination of daughter strands help in determining the DNA sequence

3' hydroxyl (OH) group

Forms phosphodiester bond between nucleotides and allows new nucleotide to be added to the existing chain

5' phosphate

The beginning of the DNA/RNA molecule that allows DNA/RNA synthesis

Why is template DNA needed for DNA sequencing?

It is needed for the polymerase to add bases to the DNA strand that is complement to the template

Why is a primer needed for DNA sequencing?

If there is no proper priming site, or the primer is not strong enough to bind with the template, the amplification of DNA is not possible

Cell lysis

The breaking down of the membrane of a cell to extract DNA, RNA, and proteins, as well as purifies organelles

Protein purification process

Use chromatography to target protein from other molecules in a cellular mixture and purify the target protein, in the chromatography system, it interacts with the mobile and stationary phase and the stronger molecule interactions stay behind while the weaker interactions elute through

IMAC (immobilized metal affinity chromatography)

A method used to purify emGFP. In the column, the his-tag protein is attracted to metal and binds to the nickel within the column, attached to the his-tag is the target protein, imidazole is added to the column to competitively bind to the nickel ions so that the his-tag and target protein together unbind from the nickel and are eluted from the column

BCA protein assay

A technique used for quantifying the total protein amount in a sample

What are 2 critical reactions in the BCA assay?

Peptide bonds that reduce copper, and the formation of a purple complex that absorbs light at wavelength 562 nm

What does the result of BCA assay show?

Under a spectrometer, the plate containing the lysate samples is read and measures the protein concentration vs. absorbance of light at 562 nm to generate a standard curve

Why are proteins measured at 280 nm for BMA assay?

Measuring at 280 nm is fast and convenient since no additional reagents or incubation is required, amino acids with aromatic rings are the reason for absorbance peak at 280 nm

SDS PAGE process

A technique that separates proteins based on size using a polycrylamine gel. SDS is used to denature the protein and line up negative charge along the protein polypeptide chain, as well as breaks cysteine disulfide disulfide bones fo aid in protein unfolding

Why is polycrylamine gel used in SDS PAGE?

It has a tighter matrix and the gel acts to hinder the movement of larger proteins, thus allowing seperation

Why is SDS PAGE sample incubated?

Incubation of the sample at 95 degrees is crucial because boiling is used to break the secondary structure of the protein and facilitate the breakage of disulfide bonds and the binding of SDS to the protein

ELISA

ELISA is an assay that is used to identify the presence of a specific antibody or antigen in a sample at very low concentrations

ELISA process

In lab, ELISA is used to determine the concentration of antigen (GFP) in the protein sample. The well plate is coated with an anti-tag AB, the capture antibody/analyte is added and then the sample and capture + detection antibodies are added which bind to the analyte and all are immobilized, the well is then washed and detection reagent + stop solution is added to see the enzyme reaction

Antibodies

Specific proteins that attach to antigens to inactivate them, keeping them from harming the body

Antibody structure

Antibodies have 2 heavy chains and 2 light chains joined by disulfide bones. It is in a Y shape with each chain having a constant and variable region. There are 2 antigen binding sites

ELISA standard curve

The generated standard curve represents the absorbance of the protein from low concentrations

HRP

The development solution that catalyzes the substrate and generate a fluorescent product

TMB

A substrate used for staining and being a visualizing reagent, used for detecting HRP activity

LFA (lateral flow assay)

A diagnostic device used to confirm the presence of absence of a target analyte

LFA structure

LFA is made of a sample pad, a conjugate pad, a nitrocellulose strip that has a test and control line, and a wicking pad

LFA process

A sample is added to the sample pad which neutralizes the sample and filters contaminants, the sample then flows to the conjugate pad of nanoparticles with antibodies, the antibodies are released and mixed with the sample, if there are target antigens that the antibody recognizes, it will bind, the mixture then goes to the test and control line to determine a positive or negative result

LFA test line

The line generates a signal correlated to the presence of antigens in the sample

LFA control line

The line generates a signal confirming the assay works

Gold nanoparticles

A signaling molecule, the sample rehydrates the signal reagent that contains another antibody conjugated to the signaling molecule

Enzymes

Molecules/catalysts that increase the rate of a reaction, they act to bind and stabilize the transition state of a reaction which increases the reaction rate by lowering activation energy

Where is the initial reaction velocity found?

It is found from the slip at t = 0 of a plot product concentration vs. time

Vmax

The maximum rate of a reaction which occurs when the enzyme is completely saturated with substrate

Km

The Michaelis-Menten constant found where the reaction rate is 1/2 Vmax

Turnover number

The max number of substrate molecules that can be converted to product by each enzyme per second