Labs Flashcards
What are the differnet micropipette volumes?
P10
P20
P200
P1000
how do you balance a centrifuge?
ensure eppendorfs full w equal volumes at opposite points to each other- if only one then use water in a ‘blank’
what is molarity?
measure of concentration of solute in solution
what are moles?
molecular weight of a substance expressed in grams
molarity = ?
moles per litre
distinguish between moles and molarity
moles- quantity of material
molarity- concentration of material in solution
what is avagadros?
one mole of substance
what calculation is used for stock solutions?
C1V1= C2V2
what is a lowry protein assay?
determine protein concentration in unknown sample
what reagent is used for colour chnage in lowry?
what is the colour change?
folins reagent
yellow/brown to blue
what is used to measure absorbance of samples in protein assays?
spectrophotometer
describe in vitro/ in vivo
in vitro- in glass
in vivo- in life
which cell type was used in tissue culture?
COS7 cells
Fibroblastic cells from monkey kidney
what terms can be used to describe splitting cells in culture?
sub culture or passage
what is passaging?
removal of media and transfer of proportion of cells into fresh gorwth medium
what are the growth phases of cell during tissue culture?
lag phase
log phase- proliferation of cells
how often were the cells cultured?
twice a week
Mon/Fri
what is PBS?
Phosphate Buffered Saline
used to wash the cells
what enzyme was used to detach the cells from the bottom of the culture dishes?
Trypsin EDTA
What growth medium was used in tissue culture?
DMEM media
what were the components of the DMEM?
10% FBS (extra nutrients)
glutamine- essential amino acid
sodium pyruvate- carbon source
1% penstrep- prevent infection
phenyl red dye- pH indicator
what is confluency?
what % is best when passaging in culture?
coverage of dish by cells
80%- as they are in the log phase of growth at this point
What in the DMEM media inhibits trypsin activty?
calcium
thsi is why cells are washed with PBS
describe a 1:5 split
2ml cell suspension into new dish + 8ml DMEM
What is GFP?
Green Flourescent Protein
What bacteria were used for culture?
E.coli
what was the broth that the bacterial colony was transferred into?
LB broth containing candymiycin
what is a plasmid?
double stranded, circular extra chomosomal DNA of bacterium
what method was used to isolate the plasmid DNA?
alkaline lysis
describe isolation of plasmid
cell growth and harvest
re-suspension (TRIS, EDTA, Glucose)
lysis (SDS, NaOH)
neutralisation (potassium acetate)
cleanign and concentration (ethanol)
what are the components of alkaline lysis buffer?
SDS detergent- cleaves phospholipid bilayer of membrane
Alkali- NaOH, denatures structural proteins of cell membrane and breaks hydrogen bonds
what was in the suspension solution for plasmid DNA?
TRIS- buffer
Glucose- osmotic pressure
EDTA- chelates divalent cations Mg+, Ca+
what does the potassium acetate do during alkaline lysis?
neutralises solution allowing hydrogen bonds between bases to re-establish
why is addition of potassium acetate important?
selective process- plasmid DNA can renature but gDNA is too big
(make sure not to vortex as this can shear gDNA into smaller bits that can reaneal)
how is double stranded plasmid DNA separated from gDNA, SDS and denatured proteins?
dissolve easily in soln whilst others stick together to form precipitate seperarated by centrifugation
what is used to seperate the DNA?
isopropanol
how is plasmid DNA cleaned (debris, EDTA, salt)?
ethanol precipitation
what is transfection?
introdunction of foreign DNA into nucleus of eukaryotic cells
what are the two types of transfectant?
stable- cells have intregratde foreign DNA
Transient- foreign DNA does not integrate in genome but genes are expressed for limited time (24-96hrs)
what methods can be used to transfect cells?
Electroporation
calcium phosphate precipitation
lipid mediated lipofection
retroviral infection
microinjection
DEAE- dextran
how does electroporation work?
membrane becomes highly permeable and more positivley charged, negative DNA crosses more easily into cell
pros/cons of electroporation?
nonchemical method (doesn’t alter biochemical structure)
easy
effeicient
cell mortality (if suboptimal conditions)
how were cells split to ensure 70% confluecny at time of transfection via electroporation?
split 1:3
what are the components of the lysis buffer for the cell lysates?
Tris HCL- buffer
Sucrose- osmotic pressure
EDTA/EGTA- protease inhibitors, chelate Mg+, Ca+
Sodium flourode/Sodium pyrophosphate- protease inhibitors
Triton X-100- detergent, permeabilises cell membrane
why were the cell lysates kept on ice?
slow down denaturation of protease
why is B-Mercaptoethanol added to cell lysates?
reduces disulphate bridges- proteins can separate on W. Blot
What are the three elements of a Western Blot?
Separation by size
trasnfer to solid support
marking of target protein
what are PAGE gels and why are they a hazard?
polyacrylamide gel electrophoresis
can contain unploymerised acrylamide which is a neurotoxin- need ot be disposed of in cytotoxic bin
what was the gradient of PAGE gel used?
4-12%
lower % of acyrlamide at top of gel and high % at bottom
the proteins have what charge when runnign W. Blot?
negatively charged
run from -ve electrode to +ve electrode
how are the proteins separated during W. Blot?
separated by weight- kDa
how long were the W. Blot gels run for?
60mins
(keep on ice to avoid overheating)
what is used for immunoblotting of the proteins follwoing western blot?
nitrocellulose membranes
electrophoresis
during electrophoresis proteins migrate from the gel onto the nitrocellulose membrane. why do they bind to the membrane?
hydrophobic interactions
why is marvel used?
masks isotopes so prevents binding of antibody to preoteins non-specifically
what is the primary antibody?
specific to GFP or GAPDH
what is the secondary antibody?
bonds to both primary antibodies
conjugated to HRP (horse radish peroxidase)
is GFP baturally found in COS7 cells?
No- ill only be expressed in those trasnfected with plasmid DNA
what was our housekeeping protein?
GAPDH, naturally found in all cells
What is ECL?
Electrochemiluminescence
what are the two solutions used to create ECL?
Hydrogen peroxidase
luminol
how does ECL emit a light signal?
luminal is oxidised when contact with HRP and so becomes excited- emits light when it falls back down to baselinedetected by x-ray film
how long does ECL last?
1 hr
what is PCR?
amplification of small segments of DNA
what is the enzyme used in PCR?
Taq Polymerase
synthesizes two new strands of DNA using originals as templates
what are the steps involved in PCR?
denaturation- 93C
annealing- 61C
elongation- 72C
what is sybersafe?
DNA gel stain that flouresces under UV light
what type of PCR was done to determien is kRAS mutation was present?
genotyping PCR
(wild type and het)
