DAT Biology - Microscopy and Lab Techniques

Fixation

sticking or securing cells onto a slide, preserving them as close lifelike state as possible, allow the stain to be applied better

staining

application og stains or dyes to a speciment to add color and contrast, often killing the specimen

Optical microspcopy

cells are viewed directky, very important that specimen is thin

whats can optical microscopy observe

live/dead cells

Stereo microscope

low magnification used to view the surface of a speciemen (3D)

Compound microscope

multiple lenses used to provide adjustable magnification, allows for viewing of simple, one cell thick samples requiring fixation and staining

poor image contrast

Whats the bad part about a compound mcroscope

There is poor image contrast and viewing of simple, one cell thick samples with fixation and staining is needed

Bright field microscope

compound microscope with a bright light

Phase contrast microscope

compound microscope allows to view samples of live unstained cells with high image contrast

Fluorence microscopy

view live cells that are tagged with fluorophores

allows cellular components to be viewed

fluorophores

chemical markers used to tag target structures

FRAP

Fluorescece recovery after photobleaching

done on large amounts of fluoresecent molecules

1. the baseline fluoresence is measured

2. an area of the sample is bleached

3. and over time, the fluorescence is measured as molecules get replaced via diffusion

electron microscopy

electrons are fired at sample, and they bounce off, causing them to pass through a magnetic field, allowing smaller objects to be viewed

specimen must be stained with a metal coating first

Whats the negatie part of electron microscopy

the specimen must be stained with a metal coating and killed

two types of electron microscopes

SEM and TEM

SEM electron microscope

scans 3d surface images

TEM

electron microscope that scans highh resolution 2D images of the internal structure of a sample

lag phase

bacterial proliferation is stagnant, cells initially are adapting to the medium

Growth rate = death rate

exponential phase

period of exponential cell growth where growth rate is more than the death rate

Stationary phase

A second period of stagenant proliferation where the growth rate is equal to the death rate

Death phase

phase in the bacterial growth curve where death rate is greater thatn the growth rate, caused by a lack of resources

Differential centrifugation

seperating solutes in a given solvent based on size and density ,

the most dense ones with the biggest size form a pellet first

Pellet

result of differntial centrifugation

componetns which have sedimented to the bottom of the tube

organelle density

1. nucleus

2. mitochondria, chloroplasts, and lysosomes

3. Er and golgi fragments

4. ribosomes and large macromolecules

Karyotyping

observing of chromosomes under microscope during metaphase

CRISPR

tech allowing for editing of specific genomic regions

with target sequences being deleted or inserted

DNA fingerprinting

an identification technique which uses noncoding DNA fragmented with restriction enzymes and analyzed

via gel electrophoresis

What is DNA fingerprinting used for

Paterity testing and forensic examination

DNA sequencing

use of PCR with DNTPS and dDNTPS, leading to the formation of DNA chains with multiple lengths, allowing a sequence to be determined

what is a dDNTP

used in DNA sequencing, a deoxynucletide that does not have the 3’ OH needed to conntinue elongation, leading to a stop in copying

PCR

a process that allows for the creation of 1 billion + copies of a DNA fragment

Occuring in denaturation, annealing, and elongation steps

Denaturation

Intense heating at around 94-95 degrees celcius seperating the DNA double strands into single strands

What is the denaturing phase in celcius

94-95 degrees celcius

What is the anealing phase in celcius

55 degrees celcius

Primer anealing phase

the sequences are cooled form 94 - 95 degrees celcius to about 55 degrees celcius, allowing DNA primers to hybridize with the single strand

What temperature is the enlongation phase at

72 degrees celcius

Elongation phase

the sequences are cooled form 55 degrees celcius to about 72 degrees celcius, allowing taq polymerase to add nucleotides to the 3’ ends of the strands

Bacterial cloning

Eukaryotic gene products are cloned using prokaryotic cells

How does the process of bacterial cloning work

1. Processed eukaryotic mRNA is converted into CDNA via reverse transcriptase

2. the CDNA is incoorporated into a plasmid via DNA ligase

3. The plasmids are incoorporated into competent Bacteria

4. The gene of interest is located via AB resistance which is attached to the target gene

What is bacterial cloning used for

medicine making

Where do restriction enzymes recognize and cut DNA at?

Palindromic sequences

Gene therapy

Medical treatment where target genes are inserted into patient cells , with viruses being the prefered vector

why are viruses the preffered vector for gene therapy

they have a high transduction rate

why are viruses a bad vector for gene therapy

the have the potential to trigger an immune response

Why are nonviral vectors bad for gene therapy

They are not as effective as viral vectors

Why are nonviral vectors good for gene therapy

they dont trigger an immune response

What are examples of nonviral vectors

CRIPSR and naked plasmid DNA

What are potential uses for gene therapy

treatment of diseases with genetic involvement

ELISA

Determine if a person has a specific antigen, where antibodies are placed on a microtiter plate with a sample from the individual, where binding leads to a color change

what is ELISA used for

Diagnoses of diseases like HIV

Pulse Chase experiments

used to observe protein movement in the cell

includes pulse and chase phase

track proteins using staining

Pulse phase of pulse chase experiments

radioactivley labeled amino acids are incoorperated into proteins

chase phase of pulse chase experiments

Prevention of readioactivley labeled protein production

What are the uses of pulse chase experiments

to study gene expression and protein fates

Gel electrophoresis

technique used to seperate DNA fragments or protein fragments by size or negative charge in an agarose gel

with negative cathode on top and positive anode on the bottom

Uses of gel electrophoresis

Genotyping, fingerprinting, and diagnostic testing

What is SDS PAGE used for

protein gel electrophoresis

What is SDS PAGE

Gel electrophoresis which uses SDS to seperate proteins based on size, charge, and density

What is SDS

Sodium dodecyl sulfate

it is a strong detergent that denatures protiens and gives them a negative charge

What are the uses for SDS PAGE

Assesing protein purity and size

SNoW DRoP

southern blotting is for DNA

northern blotting is for RNA

western blotting is for proteins

Exonucleases

nucleotide cleaving enzymes that cleave from the ends of a polynulceotide chain

What ends can exonucleases produce

sticky

Endonulceases

nucleotide cleaving enzymes that cleave from the inside of a polynulceotide chain

What ends can endonucleases produce

sticky and blunt

Restriction enzymes

Special endonucleases that mostly cut DNA at palindromic sequences

Palindromic sequences

Short regions where the sequence reads the same in the 5’-3’ on both strands

Sticky ends

Produced by endo and exonucleases

DNA fragments with single chain overhanging segments (unpaired nucleotides)

Blunt ends

Produced by endo nucleases

DNA Segements without any unpaired nucleotides

Recombinant DNA

DNA produced when DNA fragments from different sources are joined together

Southern blotting

DONE FOR DNA

Identify fragments of known DNA sequence within a large population of DNA

electrophoresized DNA is seperated into single stands, put onto a filter, and that filter is then exposed to DNA probes,

all the non probed DNA are then washed off

Northern blotting

Southern blotting but with RNA

Western blotting

Northern blotting but with SDS PAGE

and primary (bind to target protein) and secondary antiboides (multiple bind to primary antibody)

Genome

the study of all genes present in an organism’s genome and how they interact

Gene anotation

The process of identidying the location of genes and coding regions within a genome and determining each of their functions

needs genomic library and DNA microarrays

Genomic library

Storing of an organism’s genome

DNA fragments incorperated into plasmids, screened for using using antibiotic resistance and color changing techniques, cloned by bacterial cloning

DNA microarray

Contain thousands of DNA probes, bind to complemetary DNA fragments, allowing researchers to see which genes are expressed

How are DNA microarrrays perforemd

1. a cell is isoltated, and the mRNA is removed before exposing it to reverse transcriptase in atfer to produce cDNA

2. The cDNA is then hybridized with DNA probes, and the microarrays are examined for fluorescence, the microarray can now be compared with the sequenced genome

why is p