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Method Flashcards

1
Q

How does cell passaging occur?

A

Set up the protocol in a tissue culture hood - so everything can be done in a sterile environment
Sterilise by spraying ethanol

Then look under the microscope to check the cells for contamination/infection

Remove the culture medium using a sterile pipette tip

Wash the cells with PBS solution, inserting with a new pipette tip
Then discard the PBS

Replace pipette tip and add in trypsin EDTA solution
Leave for 10 minutes and allow the cells to detach

Check if the cells have detached under a microscope
Using a 5ml pipette tip, break up the clumps of cells by pipetting up and down

Add in culture medium to the cells

Take a sample and work out the cell density using a heamocytometer and counter

Based on the cell count determine the ratio the cells that need to be passaged

Remove the excess cell solution and top up with media to make up 10ml in the flask

Update passage number on flask

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2
Q

Where is the genetic material kept inside the fertilised zebrafish embryo?

A

Cell

The other areas - chorion, yolk

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3
Q

What is the mid-blastula transition?

A

When the embryo starts transcribing its own genome
Occurs at the 1000 cell stage
Until then the embryo used mRNAs deposited in the egg by the mother prior to fertilisation

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4
Q

What are the 3 layers formed during gastrulation?

A

Ectoderm (from epiblast), mesoderm, endoderm (hypoblast)

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5
Q

What are the developmental stages of the embryo?

A 
Cleavage - 1st step
Blastula - 2nd step 
Gastrula - 3rd step 
Segmentation - 4th step 
Organogenesis - 5th step
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6
Q

When does the heart start beating?

A

36-48 hpf

Can be as early 28hpf

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7
Q

When does the embryo start moving?

A

Before coming out of the chorion

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8
Q

List some other organisms can be used to understand human development

A

Mouse
Chick
Fruit fly
Nematode/frogs

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9
Q

What structures does the endoderm give rise to?

A

Gives rise to majority of digestive tract - entire epithelium

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10
Q

What structures does the ectoderm give rise to?

A

Nervous system + epidermis

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11
Q

What structures does the mesoderm give rise to?

A

Muscle cells + connective tissue

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12
Q

Movements during gastrulation

A

Epiboly
Involution
Convergent extension
Radial intercalation

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13
Q

What is the notochord?

A

Defining structure of the chordates, and has essential roles in vertebrate development
Major skeletal element of the developing embryo

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14
Q

What is the 3 tier licensing system?

A

Establishment licence - certificate of designation
Project licence - specific research/testing programme
Personal licence - specific individual/competency

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15
Q

When are the licences approved?

A

If benefit outweighs the cost
If there is no non-animal alternative
Minimum number of possible animals used
Using animals with the lowest sensitivity to pain as possible
Pain is minimised
Research premises have necessary facilities to care for animals

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16
Q

What is the local ethical review?

A

Committee of scientists and lay people, review and justify the activity and use of animals
Done for every research/testing activity

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17
Q

What is required to carry out animal research?

A

Licences and approval from LEC

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18
Q

What are the 3 Rs?

A

Replacement - use of non-animal testing methods/alternative techniques
Refinement - welfare, improve procedures, better housing
Reduction - minimum number of animals, fewer animals, more information

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19
Q

What year was the Animal Scientific Procedure Act brought in?

A

1986

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20
Q

What does ASPA 1986 do?

A

Regulates use of protected animals in experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to the animal.

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21
Q

Which animals are considered by ASPA?

A

Any living vertebrae (other than humans) and any living cephalopods

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22
Q

What does ASPA ensure? (Animal Scientific Procedures Act)

A

Animals are cared with the best standards of animal husbandry
Home office inspection system in place to ensure rules are not violated

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23
Q

What is the central dogma?

A

DNA–>RNA–>Protein

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24
Q

Which sites regulate the expression of a gene?

A

Regulatory regions

eg. promotor

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25
Q

What direction is a sense strand?

A

5’ to 3’

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26
Q

Describe transcription

A

Transcription = unwinding DNA double helix, exposes anti-sense strand
Allows RNA pol to bind
Transcribe RNA in 5’->3’ direction (using anti-sense strand as a template)
generates RNA in sense orientation (5’ left, 3’ right)

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27
Q

When does transcription occur?

A

Genomic DNA transcribed in cells where the regulatory regions are activated

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28
Q

What happens after RNA is transcribed?

A

Processed, introns removed, 5’end modified, 3’ poly A tail = mature mRNA
Exported into cytoplasm
Serves as template for translation

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29
Q

Briefly describe the process of translation

A

mRNA positioned within the ribosome (between 2 subunits)

tRNA with amino acid attached, recognises codon, binds and allows polypeptide chain to grow

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30
Q

What do expression pattern of genes allow?

A

Give ideas about function of gene

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31
Q

How can the expression pattern of genes be visualised?

A

Staining techniques
In-situ hybridisation - localisation of mRNA within tissue
Immunostaining

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32
Q

What is in-situ hybridisation?

A

Looks at mRNA
Recognised using a specific RNA probe
Probe has DIG (digoxigenin)

Probe hybridised overnight to embryo
PBS washing to remove anything not specifically bound to mRNA

Incubate embryo with Ab recognises + binds to DIG label on probe

Ab has enzyme attached (Alkaline phosphatase)
Wash again

Add substrate for AP (BCIP/NBT) undergoes a colour reaction turns it into a coloured precipitate (dark purple)

Gives staining visible in regions contain mRNA

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33
Q

What does in-situ hybridisation show?

A

Gives staining visible in regions contain mRNA

shows how much mRNA is present - approx

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34
Q

How else can in-situ hybridisation be carried out (without enzyme linked Ab system)?

A

Fluorescently label Ab, detect by fluorescence

= FISH

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35
Q

What is immunostaining?

A

Looks at gene expression by looking at protein

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36
Q

Describe immunostaining

A

Generate Ab that recognises Ag (a protein in cell/tissue)
Amplify the signal using a secondary Ab which recognises the primary Ab (Ig domain)
NB. secondary Ab is generic for Ig
primary Ab is specific to the protein itself

2nd Ab can have AP attached - will change colour when add a substrate
Can also be fluorescent

Can combine different Ab and different colours on one specimen + look at 2 or more proteins at a time

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37
Q

What colours are fluorescein and rhodamine?

A 
Fluorescein = green 
Rhodamine = red
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38
Q

How is a probe, to recognise mRNA in tissue, made?

A

Make cDNA from mRNA using reverse transcription to generate ssDNA
Then make dsDNA + amplify by PCR = cDNA

c DNA (ds) used to generate anti-sense RNA complimentary to sense mRNA that want to detect present within the sample

Antisense RNA has DIG attached (probe)
Incubate with embryos
Able to detect where probe binds to specifically + mRNA accumulation within the cell

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39
Q

Probe production in more detail

A

mRNA want to detect made by the cell from DNA in cell
mRNA processed by cell + has poly-A tail attached
This is the sense strand of RNA

cDNA made through reverse transcription
Oligonucleotide first binds to mRNA- short stretch of nucleotides contain lots of Ts which are complementary to polyA tail on sense strand

in reaxn Ts will bind to As on sense strand
Serve as site where reverse transcriptase can bind to end of Ts and synthesise copy DNA in 3’ to 5’ direction

Synthesises cDNA in 3’->5’ direction
produces anti-sense cDNA

cDNA amplified by PCR to produce ds DNA
uses specific primers

This is then separated where the sense strand is used to produce anti-sense RNA which is the probe

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40
Q

What is the point of the specific primers in the PCR amplification?

A

Amplifies just the single gene that interested in from a mixture of cDNAs as process of binding Ts to As not specific to any one gene
PCR primers used are specific for the one gene interested in

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41
Q

What else is done when designing the PCR primer?

A

Another RNA pol binding site added onto end of anti-sense strand DNA = T3
This is required to generate an RNA probe for in-situ hybridisation

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42
Q

What happens after ds cDNA made?

A

RNA pol separates 2 strands
uses sense strand as a template and produces antisense strand - complementary to sense strand mRNA present in tissue
This is the RNA probe

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43
Q

What modification is made to the nucleotides when the antisense RNA probe is being made?

A

Nucleotides used have DIG attached

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44
Q

How would in-situ hybridisation in a zebrafish work?

A

Have mRNA want to detect expression of in zebrafish
hybridise probe to mRNA
(probe = antisense RNA strand with DIG attached)
wash away anything not bound specifically
incubate with Ab bind to DIG
Ab have AP attached - add substrate
generates a purple colour within the embryo

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45
Q

What is trypan blue staining for?

A

Diazo dye used to colour dead tissues or cells selectively

Gives and idea of health of cells and percentage viability

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46
Q

What is the basis of trypan blue staining?

A

Based on negative charge - won’t interact with cell membranes unless they’re damaged
Living cells exclude the blue dye and appear bright under microscope
Dead cells take up dye and appear blue

Readily available, inexpensive and rapid

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47
Q

What is an MTT assay + what is its basis?

A

Colorimetric assay for assessing metabolic activity
NADPH dependent cellular oxido-reductase enzymes may reflect the number of viable cells present
Enzymes can reduce MTT to form purple, insoluble formazan crystals

Increased purple colouring = increased number of viable cells
Used in 96 well plates

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48
Q

Describe the process of bacterial transformation

A

Where bacteria take up a foreign genetic material (DNA plasmids) from the environment
These cells are known as competent cells

Plasmid vector with PCR DNA inserted is inserted into competent bacteria through electroporation or chemical transfer
Chem transfer = lipofection
or incubate with CaPO4 - has DNA associated and is insoluble in water, will precipitate
This is then taken up by endocytosis by bacteria

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49
Q

How is DNA (from pCR) inserted into a plasmid?

A

Through TA cloning
During amplification using Taq polymerase, it adds on an A at the end of the PCR product (3’end)

Plasmid vectors have T overhangs (3’) where they have been cut to be linearised

Are complementary to each other and A+T will form weak H-bonds
Hold structures in place, allowing DNA ligase to form phosphodiester bonds between them (ligating the DNA +vector together)

It also prevents the recircularisation of empty vector as T on one strand is not complementary to T on another strand

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50
Q

How are colonies selected for the inserted DNA?

A

Colonies are grown on selective media - ampicillin
to select bacteria have taken up the plasmid

If the DNA has been successfully inserted in the plasmid, it will destroy the lac-Z gene
Beta galactocidase enzyme won’t be produced by these bacteria and so when substrate X-gal added will not convert into blue product
Successfully transformed colonies = white colonies
Blue ones are unsuccessful

Activity of lacZ gene indicates whether DNA insert incorporated into plasmid or not

Prevents unnecessary screening of colonies - can just pick those that indicate have incorporated desired gene

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51
Q

How can desired DNA be extracted from bacterial cells?

A

Using a miniprep protocol + spin column
Put spin column in ependorf tube + spin in centrifuge
Wash
DNA will stick to resin on tube
everything else washed away + elute clean DNA from column itself

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52
Q

What is the purpose of bacterial transformation?

A

To produce multiple copies of the DNA

Don’t need to do PCR again, just go to stocks of bacteria bacteria

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53
Q

What are the principles of a Bradford assay?

A

Colorimetric assay based on absorbance shift of dye Coomassie Brilliant blue
Protein in assay = blue
No protein in assay = brown

Because in acidic conditions dye is converted from red to blue and will bind to the protein being assayed

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54
Q

What are the advantages of using zebrafish in research?

A

Genome similarities with humans
Embryos develop outside the mother’s body - easier to study early development
Grow at fast rate
Cheaper than mice
Embryos are transparent - can see development of internal structures
Hundreds of offspring at weekly intervals

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55
Q

Advantages of mice in research?

A

Similar to human genome
Easy to maintain - small so easy to house
Have short life cycle
Cheap
Relatively easy to manipulate genome - add/remove genes, to better understand role in body
Help study complex biological systems - immune system, nervous system

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56
Q

What is brightfield microscopy?

A

Sample attenuates light passing through it
Field outside the sample bright
Dye required in sample to attenuate light through it

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57
Q

+ve/-ve brightfield microscopy?

A

+ve - useful for things can stain + staining sections

-ve - can’t use for things can’t stain eg. live cell need something else

58
Q

How does reverse transcription occur?

First strand cDNA synthesis

A

Requires mRNA, RT enzyme, primer + dNTPs

RNA incubated with primer at 70 degrees to denature RNA secondary structure

Then chilled on ice to allow primer to anneal onto RNA

RT, dNTP + buffer added to reaction

Then temperature changed to 37 degrees or higher to allow transcription to occur – 1hr

Then heated again above 70 degrees to denature enzyme = cDNA

59
Q

How is the specificity increased?

A

Can generate gene specific primers - only make cDNA for specific target
yield is lower because targeting 1 transcript

60
Q

How does PCR occur?

A

Heat up to 95 degrees and denature DNA – then do annealing stage by bringing temp down to 55-60 degrees, allows primers to bind to DNA

Heat again to 72 degrees to allow the polymerase to add the nucleotides starting from the primer + extend the molecule

This process is repeated multiple times to amplify the target DNA

Copies of target DNA will increase with each cycle, as will copies of variable length DNA fragments (but are a much lower proportion)

61
Q

What must happen after a PCR reaction?

A

DNA must be purified - using gel electrophoresis
Separates mixed DNA fragments to estimate size or isolate fragments
And do DNA clean up protocol + elute DNA (spin column)

62
Q

Give a brief outline of the steps necessary to generate double-stranded cDNA for a single gene, starting from a piece of tissue that is known to express the gene of interest.

A

Extract the mRNA from the tissue – using spin column and miniprep test kit
Then produce cDNA by putting the mRNA with random primers, RT enzyme, dNTPs and carry out reverse transcriptions – first strand cDNA synthesis

63
Q

What is Western blotting used for?

A

To detect specific proteins in a sample of tissue extract

Identify specific proteins from a mixture of proteins extracted from cells

64
Q

What happens in Western blotting?

A

Sample undergoes protein denaturation, followed by gel electrophoresis (to separate by size), visualisation (actual western blot) where mark target protein using primary and secondary antibody to visualise

65
Q

Describe gel electrophoresis used in Western blotting

A

Electrophoresis is done using 2 gels –

Stacking gel pH6.8 (more acidic), separates protein poorly but allows form thin, sharply defined bands

Separating gel, pH8.8, pores are narrower, so protein is separated by their size more in this – smaller proteins travel more easily + rapidly than larger proteins

Proteins will travel towards positive electrode when voltage is applied

66
Q

What does denaturation fo the protein allow?

A

Denaturing the high structure ensures that negative charge of amino acids is not neutralised, enabling the protein to move in an electric field – applied during electro-transfer

67
Q

What happens after the gel has run?

A

After separation proteins are transferred to membrane through electric field oriented perpendicular to surface of gel

Semi-dry blotting

Transfer protein from gel onto PBDF membrane by using semi-dry blotter
Sandwich membrane with gel on top in between Filter paper
Clamp between 2 electrodes in blotter
Apply electrical current - protein within gel will migrate onto membrane
Fix protein onto membrane

68
Q

How is the Western blot produced?

A

Introduce primary Ab to membrane, Ab recognises epitope to protein of interest
Then introduce secondary labelled Ab
2’ Ab labelled with Horseradish peroxidase

Incubate + wash off unbound Ab (PBS)

Add in substrate
Interaction between HRP + substrate allows light to be emitted so gives chemiluminescent signal

69
Q

How can the light signal be visualised from the Western blot?

A

Visualise on X-ray film by exposure for few seconds/minutes

Should place x-ray film after reaxn occurs - get bands forming

70
Q

What does HRP do?

A

In presence of hydrogen peroxide, converts luminol to intermediate dianion
Dianion emits light on return to ground state

71
Q

For how long does light emission continue after exposure to substrate?

A

Maximal immediately after exposure

Continues 0.5-1hr after

72
Q

What are some limitations of Western blotting/semi-dry blot?

A

Data produced is semi-quantitative – relative comparison of protein levels but not an absolute measure of quantity
Variations in loading + transfer rates between samples in separate lanes which are different between different blots
Need to be standardised before precise comparison can be made
Signal generated is not linear across concentrations in a range of samples - can’t be used to model the concentration

73
Q

Why should they be transferred onto a membrane?

A

This enables incubation with different Ab allows pick up specific proteins within the extract
As up until that point there are a mix of proteins within the extract - helps quantitate specific levels of proteins within the extract

74
Q

How do Bradford assays quantitate a reaxn?

A

Put the plate with the bradford assay in a plate reader + read at 595nm
Allows production of standard curve
From it can calculate amount of protein within a sample

75
Q

How is the position of the ion in the gel determined in SDS-polyacrilamide gel electrophoresis?

A

Discontinuous gel system - where separating gel has different pH to stacking gel
Running buffer has glycine which has Hydrogen side chain
Position of ion dictated by pH hydrogen is running in

Stacking gel allows protein stack at fine line at the interface
Once protein enters separating gel - can be separated according to molecular weight

76
Q

What components of the sample buffer help with loading?

A 
Bromophenol blue
Glycerol 
SDS
Beta mercaptoethanol 
\+ sample buffer must be at right volume
77
Q

What is the function of bromophenol blue?

A

Allows visualisation of sample, if only used this sample would float out of the wells

78
Q

What is the function of beta mercaptoethanol?

A

Redcuing agent + reduces disulfide bonds in proteins - important to load samples onto system + allow it to migrate

79
Q

What is the function of glycerol?

A

Adds density, so sample don’t float out of wells

80
Q

What is the function of SDS?

A

Anionic detergent - breaks down non-covalent bonds
(as protein structure has lots of bonds - H bonds, ionic bonds)

Binds to amino acid side chains, giving protein net negative charge
important to separate proteins in the system so it can migrate to the positive anode

81
Q

How to know when to stop running the gel?

A

Before loading, load a rainbow marker - mix of labelled proteins in different colours each corresponding to a different weight
Will know when to stop because know how big the molecules are

82
Q

What can stained gels be use for?

A

Can stain all the protein in the extract - shows if sample equally loaded
does not identify specific proteins + difficult to quantitate

Equal loading of protein is required to be able to compare the wells

83
Q

What are some positives of a semidry blot?

A

Semiquantitative - use software to quantitate bands gives an indication of whether there is an increase in the level of the protein or alteration in phosphorylation state
Can identify specific proteins w/extract
Can re-probe the blot - re-incubate membrane with another Ab to house keeping gene
eg. tubulin/gap-DH to show if have equal protein as they are proteins that shouldn’t be changing very much

84
Q

What are the COSH regulations and how do they maintain safety?

A

Control of substances hazardous to health 2002 - COSH
Need to fill in the forms
Allow control of substances hazardous to health and prevent or reduce exposure to substance by:

Finding out what hazards are
Risk assessment - deciding how to prevent harm
Providing control measures to reduce harm to health and ensuring they are used
Providing information, instruction and training for employees
Planning for emergencies

85
Q

What are adherent cells?

A

They are grown in a monolayer
and can be released from dish/flask enzymatically/mechanically
Growth is limited by surface area

86
Q

What are non-adherent/suspension cells?

A

Grow freely in liquid as single cells/free floating clumps

Can grow to high density in very small volume

87
Q

What is density with regard to cell culture?

A

Measure of proliferation

When gets too dense for flask, must be passaged (sub-cultured)

88
Q

What is confluency?

A

Combined with density - estimate or counted percentage of dish/flask covered with cells
100% entirely covered
10% confluency = 10% surface covered
Sub confluent level = 30-40% allows space for doubling up

89
Q

What is passaging and what is the passage number?

A

Passaging/subculture is lifting cells, diluting the volume and removing cells (enzymatically) .˙. maintaining the stock of growing cells
Passage number = number of times cell culture has been subcultured - some cell lines only grow for finite number passages

90
Q

Describe enzymatic removal of cells

A

Use trypsin-EDTA
Trypsin cleaves peptide bonds in fibronectin of extracellular matrix (would attach cells to surface/plastic)
EDTA chelates Ca in media
Ca would normally inhibit trypsin

91
Q

What is the danger of using trypsin for too long?

A

Can reduce cell viability
Can alter phosphorylation state some proteins found at the cell surface
(if looking at phosphorylation state, should transfer cells into centrifuge tube and produce cell pellet - can lyse pellet)

92
Q

How are cells mechanically removed?

A

Using a cell scraper

93
Q

What happens once the cells are detached?

A

Quantitated using a haemocytometer to determine cell density
Count cells in outermost 4 squares + use formula to work out how many cells/ml
Once know cell density/number can seed cells for experiment
If want to compare experiments, should seed cells at same number/density each time

94
Q

What can cell number be used for?

A

To determine how well cells grow - enables plot growth curve

95
Q

How are proteins isolated from a cell and where can they be extracted from?

A

Use detergents to isolate protein
Different strength of detergent isolate from different regions eg. nucleus/cytoplasm

Whole cell extract - lyse cell + isolate cell extract
Cytoplasmic cell extract
Nuclear cell extracts

96
Q

What are the different types drug screens and what type of research are they an example of?

A

Examples of exploratory research
Phenotypic drug screens:

morphological - identifies compounds that lead to a particular morphological effect

Therapeutic - identify compounds that ameliorate a disease phenotype
use model organisms where human disease features are reproduced, treat with compounds and see which are reverting disease phenotype - can be further developed into therapeutic treatment

pathway directed - know that in a disease particular signalling pathway is affected, want to find compounds would target the pathway
identify compounds that interfere/modify the activity of specific molecules/signalling pathways

give data or tools that can use for hypothesis driven research

97
Q

What is exploratory research?

A

Increase knowledge which can lead to an experiment

Not driven by initial hypothesis

98
Q

What type of research is used in labs?

A

A mix of hypothesis and exploratory research

99
Q

What is hypothesis driven research?

A

Observe a phenomenon/identify problem
Generate hypothesis - answer to question, idea as to what happens
Design experiments to test hypothesis
Allows acceptance or rejection hypothesis

100
Q

How is protein synthesis regulated in the body?

A

Regulated at 2 levels
Transcript
Global regulation

101
Q

Describe regulation at the transcript level

A

Message/transcript - as eukaryotic mRNA has innate characteristics which influence how easily transcript is translated by ribosome
eg. 5’UTR has many secondary structures which can inhibit/affect the efficiency that the ribosome scans through 5’ UTR before reaching the start codon

102
Q

Describe global regulation

A

Affects whole process within the cell
eg. tissue culture being grown in an artificial environment cells are extremely sensitive to that environment
Allow them to be overcrowded .˙. confluency is too high, allows them to be stressed
Will begin to shut down protein synthesis

103
Q

What are some factors that cause cell stress?

A 
Withdrawal of nutrients 
Overcrowding 
Temp shock
Starve by removing amino acids from tissue culture media 
Hypoxia 
Viral infection
104
Q

What happens as a result of cell stress?

A

Shut down of protein synthesis

If stress prolonged, cell will activate other pathways eg. apoptosis

105
Q

Describe translation

A

Very fast process - to supply protein in cell
Can link 400 aa in 20 seconds
Slow enough to avoid too many errors
Error rate = 1 in 10^4 incorrect amino acids

106
Q

What is peptidyl transferase activity associated with?

A

Associated with 60s subunit

links aa together during elongation

107
Q

When is the potential to initiate protein synthesis the greatest?

A

When the 2 ribosomal subunits are dissociated

As 40s needs to be trapped by eukaryotic initiation factor will allow it to enter process of initiation

108
Q

What is the principle behind confocal microscopy?

A

Laser light is focused onto defined spot at a specific depth within the sample

This causes emission of fluorescent light at exactly this point

A pinhole inside the optical pathway cuts off out-of-focus signals allowing only fluorescence signals from the illuminated spot to enter the light detector

Scanned in a raster pattern, producing imaged in a single optical plane

109
Q

How can 3D objects be visualised using confocal microscopy?

A

Scan multiple optic planes + stack using a suitable microscopy deconvolution software

110
Q

What else is possible with confocal microscopy?

A

Analysis of multicolour immunofluorescence stainings using state-of-art confocal microscope that include several lasers + emission/excitation filters

111
Q

What is confocal microscopy used to resolve?

A

Detailed structure of specific objects within the cell
Components of living or fixed cells can be specifically labelled using immunofluorescence and then visualised in high resolution

112
Q

What is a positive of confocal microscopy?

A

Produces sharp images of the exact plane of focus, w/o any disturbing light from the background or other regions of the specimen
Thicker objects can be visualised
3D structures can be visualised

113
Q

What is a limitation of confocal microscopy?

A

Sample penetration depth is limited - thicker specimens should be imaged using two-photon microscopy/LFSM

114
Q

What is widefield fluorescent microscopy used for?

A

For the detection of specific structures, molecules, or proteins within a cell
Almost any component of living/fixed cells can be stained + specifically imaged

115
Q

What type of assays are widely analysed using widefield fluorescence microscopy?

A

Immunofluorescence assays

116
Q

What else can be done with widefield fluorescence microscopy?

A

Can visualise several structures in parallel, which then appear as different pseudocolours in the obtained image

117
Q

What is the principle of widefield fluorescence microscopy?

A

Based off of light microscopy

Fluorophore coupled Ab will bind to the specific structures within the cell
Specimen illuminated at an excitation wavelength and viewed through a filter only allowing the emitted wavelength through

Because when illuminated the fluorophore will absorb the light at a specific wavelength and then emitted at higher wavelength

The background will be dark and structures with bound fluorophore will emit light, indicating presence of structure of interest

118
Q

What are some limitations to widefield fluorescent microscopy?

A

Whole specimen exposed to light - fluorecence signals from all focal planes are detected, leads to lower contrast in thick samples like spheroids + tissue

119
Q

When is widefield microscopy best applied?

A

Thin specimens with low background autofluorescence

eg. adherent cells

120
Q

What colour is phalloidin and DAPI?

A

Phalloidin - green, F-actin

DAPI - blue, stains nuclear DNA

121
Q

How is cross hybridisation decreased in in-situ hybridisation?

A

Using a high hybridisation temperature + formamide in HM buffer ensures high stringency of hybridisation

122
Q

How is non-specific hybridisation of the probe avoided?

A

Using high stringency washes - 0.2x SSC at 70degrees

For single locus/large probes - temperature should be higher (65 degrees) + stringency high (SSC below 0.5)

Washing for longer times can wash away more excess probe - too long may risk washing off too much hybridised probe

123
Q

What does Accutase do?

A

Similar to trypsin EDTA - allows for detachment of cells from surfaces
Auto-inhibits at 37 degrees without need for neutralising solution

124
Q

How are proteins extracted for western blotting?

A

Remove the cells needed by mechanically scraping (+use the media as it may contain cells)

Centrifuge at 4 degrees for 10 mins - discard supernatant

Re-suspend pellet in 1ml of PBS + transfer to labelled eppendorf tube

Spin down at 11,000 rm for 10 seconds - discard the supernatant

Re-suspend in 35ul of lysis buffer, pipette up+down to break up pellet

Vortex (5sec) + incubate on ice
Then sonicate 3 times to break open cells (using sound energy)

Spin in chill spinner at 14,000g for 10 minutes at 4 degrees

Transfer supernatant into new labelled eppendorf tube

125
Q

What is the role of BSA in Bradford assay?

A

BSA is used as a standard for the Bradford assay, and a calibration curve based on the mass concentration of BSA used to determine unknown concentrations of a protein.

126
Q

What is a biotinylated marker?

A

Biotinylated protein ladder detection pack is designed to detect the molecular weight ladders on Western blots when using the horseradish peroxidase (HRP) based Western detection system.

The pack contains Biotinylated Protein Ladder and Anti-biotin, HRP-linked Antibody

127
Q

What is the purpose of TEMED and Ammonium Persulphate in Western blotting?

A

Both are used to catalyse acrylamide gel polymerisation when preparing gels for gel electrophoresis

128
Q

How are proteins stacked and then separated?

A

Stacking gel is usually formulated at pH 6.7–6.8.
At this pH, ionized chloride ions migrate rapidly, raising the pH behind them and creating a voltage gradient with a zone of low conductivity

This causes glycine (from the running buffer) to ionize and migrate behind the chloride front. Most peptides in the sample, which have a negative charge due to the bound SDS, migrate between the chloride and glycine, forming a narrow band and thus becoming “stacked”.

Once the stack reaches the resolving gel, which is at a higher pH (typically pH 8.7–8.8), the increased ionization of the glycine causes it to accelerate and overtake the peptides. Smaller pore size of the resolving gel starts to have a sieving effect, resulting in the separation of peptides by size.

Most western blotting protocols use a Tris buffer of low ionic strength for protein transfer. The transfer time depends on the type of blotting apparatus and the peptide size range of interest.

129
Q

How are proteins stacked and then separated?

Know the purpose of Tris- HCL

A

Stacking gel is usually formulated at pH 6.7–6.8.
At this pH, ionized chloride ions migrate rapidly, raising the pH behind them and creating a voltage gradient with a zone of low conductivity

This causes glycine (from the running buffer) to ionize and migrate behind the chloride front. Most peptides in the sample, which have a negative charge due to the bound SDS, migrate between the chloride and glycine, forming a narrow band and thus becoming “stacked”.

Once the stack reaches the resolving gel, which is at a higher pH (typically pH 8.7–8.8), the increased ionization of the glycine causes it to accelerate and overtake the peptides. Smaller pore size of the resolving gel starts to have a sieving effect, resulting in the separation of peptides by size.

Most western blotting protocols use a Tris buffer of low ionic strength for protein transfer. The transfer time depends on the type of blotting apparatus and the peptide size range of interest.

130
Q

What is LiCl used for in riboprobe preparation?

A

Precipitation of the RNA probe

131
Q

What is PFA (paraformaldehyde) in in-situ hybridisation?

A

Used to fix cells

132
Q

What is methanol used for in in-situ hybridisation?

A

Dehydrate the cells (to allow storage?)

133
Q

What is proteinase-K used for in in-situ hybridisation?

A

Embryos are prepared for WISH by treatment to ensure that the probe will reach even the least accessible parts of the embryo.
This is most critical to ensure accessibility in zebrafish embryos that are 24hpf or older

134
Q

What is Hyb+/Hyb-used for in in-situ hybridisation?

A

Hyb+: 5XSSC, 50%formamide, 0.1%triton, 5mg/ml torula RNA, 50ug/ml heparin

Hyb-: 5XSSC, 50%formamide,0.1%triton (at 68°C)

135
Q

What is SSC used for in in-situ hybridisation?

A

Saline-sodium citrate (SSC) buffer is used as a hybridization buffer, to control stringency for washing steps in protocols

136
Q

What is triton used for in the preparation of the staining buffer in in-situ hybridisation?

A

Permeabilises the cells/tissue to allow them to stain

137
Q

Role of glycerol in in-situ hybridisation

A

For storage

138
Q

What is Ethidium bromide used for in DNA gel-electrophoresis?

A

Binds to DNA + fluoresces when exposed to UV rays

139
Q

What is IPTG used for?

A

It functions as an inducer of galactosidase activity by binding to and inhibiting the repressor. It is utilised for the induction of expression from the lac promoter and derivates.

eg. Blue/white colony selection is often used to increase the likelihood of recovering desired DNA constructs following ligation into a plasmid vector. What factor is responsible for the induction of beta-galactosidase protein in this process?
A = IPTG

140
Q

What is X-gal?

A

X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA.

Converted into blue product if lac-Z gene intact and B-galactocidase is made to convert it

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