Both CB and G lab

Carbohydrates, Lipids, Proteins, Nucleic acids

The Four classes of macro molecules

Monosaccharide

Carbohydrate monomer

Polymers

Lipids do not form

Fatty acid

lipid monomer

amino acid

Protein monomer

Creates bond and produces water as a product

Dehydration reaction

Uses water to break bond

Hydrolysis

CnH2nOn

General Glucose structure

Philic Head: Carboxyl group, Phobic tail: hydrocarbon chain

Parts of fatty acid

\

Draw glycosidic bond

1 = Monosaccharide, 2 = Disaccharide, 3-10 = Oligosaccharide, Many = polysaccharide

\# of sugars = ??????

Cellulose, Chitin, Starch

Three examples of a polysaccharides

Has both a hydrophobic and hydrophilic region

Amphipathic

Strach, Cellulose, and Chitin

Examples of Polysaccharides

Unsaturated has a C=C bond known as a kink

Saturated vs Unsaturated

A-T: 2

G-C: 3

\# of hydrogen bonds between A-T and G-C

Purine

Double ring nucleotide =

Pyrimidine

Single ring nucleotide

\

Draw an amino acid

\

Draw a peptide bond

0\.5-10, 1-20

P10 and P20 volume range

20-200, 200-1000

P200 and P1000 volume range

D = M/V

Density equation

Not expressed in mRNA

Intron

expressed and codes for protein

Exon

Does not code for proteins but does regulate gene expression

Noncoding DNA

Codes for protein

Coding DNA

short repetitive interspersed elements

SINE

Alu 1 restriction enzyme recognition site

Alu name comes from

C16 pv92 and 300bps long

Alu location and bp size

Has - chalex beads that grab metal ions like Mg2+ that are required as cofactors for enzyme like DNase (in Lysosome) which breaks down DNA.

PV92: Instagene’s purpose

In vitro, Amplyfies section of DNA

PCR is a ______ technique that ______

Kary Miulus

PCR was invented by?

Forward primer

PCR: 5’ to 3’

Reverse primer

PCR: 3’ to 5’

Denaturation: 1 min at 90-95 C, separates DNA strands

Annealing: 45 seconds at 50-55 C primers attach and make complementary strands

Extension: 2 minutes at 75 and DNA polymerase adds dNTP’s to the strand

Three steps of PCR and simple explaination

Taq, Yellowstone, *Thermus aquatics*

PCR: DNA polymerase is known as _________ and is acquired from?

Size only

Agarose Gel Electrophoresis differentiates using __________. In DNA

Cathode

DNA is attracted to which part of the electrophoresis machine

Fred Sanger

Sanger Sequencing was invented by ________

with colored ddNTP’s which lack a free 3’ OH

How are things labeled

Target DNA, 2 Primers, DNA polymerase, nucleotides (dNTP’s), Mg 2. 5 Total

Ingredients of PCR

TBE: High capacity but slow migration and lower than 2kb, TAE: fast migration and higher than 2kb but low capacity.

TBE vs TAE

encodes for green fluorescent protein

pGLO component: GFP

Origin of replication

pGLO component: ori

β-lactamase which breaks down ampicillin

pGLO component: bla

Promotes RNA polymerase when bound to AraC-arabinose

pGLO component: pBAD promoter

codes for protein that binds to pBAD promoter which additionally need arabinose and then expressed GFP

pGLO component: *araC*

taking genetic material from the environment

transformation

Foreign genetic material given by a virus or viral vector

transduction

genetic material transfer from bacteria to bacteria through direct contact

conjugation

foreign DNA introduce to eu.by physical and chemical means in a lab

transfection

growing using phenotype to distinguish

Screening

Allows growth only for selected bacteria

Selection

Remove alkaline environment allowing plasmid renaturation

What is the purpose of the neutralization buffer?

260

DNA absorbs light at which wavelength?

ampicillin

To ensure that only  the bacteria transformed with the plasmid grew in the culture media, the bacteria were grown in the presence of ____________

beer lamberts law that concentration is directly linked to concentration

The principle of Nanodrop is 

destabilizing proteins and nucleic acids

What is the purpose of lysis buffer during plasmid isolation

Attract target DNA

What is the purpose of the silica column?

Identification/characterization of gene causing a mutant phenotype observes the phenotype first and is unbiased.

forward genetics

observation of phenotype by purposefully disrupting a gene

reverse genetic

calcium chloride treatment causing plasmid to attach to the cell wall, heating in a water bath to open pores to allow entry

pGLO transfer process

Gel red

What chemical is used in lab to visualize the DNA in an agarose gel under UV light?

Differentiate between G- and G+ microbes

Principle of a gram stain

Lps, Outer membrane, thin peptioglycan layer, inner membrane

G- Bacteria Structure

pink

G- color after stain

Thick peptidoglycan layer and plasmas membrane

G+ Bacteria structure

Purple

G+ color

stains them purple, 30-60 sec

Crystal Violet

Attaches the crystal violet to cell wall, 30-60 sec

Iodine

Washes stain from G- walls, 3 sec

Decolorizor

stains g- pinks, 30 seconds

safranin

Maintaining pure culture + isolating from a mixed culture

Principle of Bacterial Streaking

Dillutuion of the \# microbes within a volume

Principle of Serial Dilution

1/10

1 mL into 9 mL dilution factor?

1/6

5 mL into 25 mL dilution factor?

1/400

Current Dilution factor: 1/4 Previous Dilution factors: 1/2, 1/5/ 1/10 What is the final dilution factor?

Salt contamination, 1.8 for DNA and 2.0 for RNA

Nanodrop: 260/230 ratio meaning and ideal value(s)

Protein contamination, ~2.0 lower than 1.8 is heavy contamination

Nanodrop: 260/280 ratio meaning and ideal value(s)

A sequence that is read the same backwards as forwards

What is a palindromic sequence

Enzyme that does not produce a overhang

Restriction Digestion: Blunt

double stranded DNA

Restriction enzymes only cut ________

A natural defense in bacteria against bacteriophages

Restriction enzyme acts as?

Annex V: stains Phosphatidylserine (PS) which is located in the cytosolic part of the membrane

Propidium Iodide: stains DNA and can only enter if the membrane integrity is low

FC: Apoptosis Staining

Necrosis

FC: Apoptosis Staining PI only

Early Apoptosis

FC: Apoptosis Staining Annex V only

Late Apoptosis

Flow Cytometry: Apoptosis Staining both

Three, Fluidics, Optics, Electronics

Flow cytometer is composed of **[A]** main subsystems. These are **[B]**, **[C]** and **[D]**.

0\.2-50 µm

What is the range of size of cells of particles that is suitable for flow cytometric analysis?

Route the laser beams to the flow cell for interrogation

FC: The purpose of the excitation optics is to:

Decrease the flow rate or the sample pressure

FC:What can be done to improve data resolution?

Maintain the sample core in the center of the sheath fluid

FC: Hydrodynamic focusing is used to

sample Core

FC: What is the name given to the portion of the fluid stream where the cells are located? 

places them in a single file line

FC: The purpose of the fluidics system in a flow cytometer is?

Sample core, Fluidics

FC: The particle suspension is injected into _________________ within the _________________.  

light

FC: When fluorescent compounds absorb light energy and then release excess energy, they emit ____________________. 

Cell Size

FC: FSC is proportional to:

complexity or granularity

FC: SSC is proportional to the ___________ or _____________ of the cell

Forward, Inside

FC: Correlated measurements of both _______________ and _____________ can allow differentiation of cells types in a heterogeneous cell population.