Common Lab Techniques Flashcards
describe how and SDS page works
add SDS to give same negative charge to proteins
driven through gel with the mass being the only factor in migration ie. larger proteins travel slower
what is a western blot
use to detect specific PROTEINS in a sample based on ability to bind specific antibodies
how is western blot performed
proteins are separated by SDS page gel electrophoresis
proteins are transferred to a sheet of nitrocellulose blotting paper
an antibody is added to the solution which binds to the specific protein of interest
the antibody has an enzyme or second antibody attached to it
the bound antibody location is revealed by incubating it with a colourless substrate that the enzyme or secondary antibody converts to a coloured product
gel electrophoresis separates dna molecules based on
size
describe how gel electrophoresis works
dna is negatively charged and when a external potential is applied dna in a agarose gel will migrate towards the positive anode
smaller fragments of dna migrate faster and further down the gel than larger fragments
what are restriction enzymes and what do they produce
used to cut specific nucleotide sequences at restriction sites
sequences are palindromic (read the same in the forward and reverse) and are 4-6 nucleotides long
sticky ends may or may not be produced
what is a southern blot used for
detect specific DNA sequence
how does southern plot work
DNA sample is chopped into smaller fragments by restriction enzymes which must not cut anywhere within the target DNA sequence
digested DNA undergoes electrophoresis, chemical denaturing with NaOH, and transferring onto nitrocellulose paper
it is then subjected to a labeled DNA probe which can only hybridize to the DNA fragment of interest
what does polymerase chain reaction do
amplify a single piece of DNA generating millions of copies of a particular DNA sequence
what are the key reagents of PCR
DNA template
2 DNA primers complementary to 3’ ends of sense and anti-sense strands of DNA target
Taq polymerase to act as DNA polymerase
dNTPs which act as building blocks to synthesize new DNA strands
what are the 3 steps in the PCR process
- denaturation: 90 degrees, DNA melts to single stranded molcules
- annealing: 50 degrees, primers anneal upstream and downstream of the target sequence
- elongation: 70 degrees, tan adds to template strand
repeat 30 times
4 differences from PCR and DNA replication
PCR in vitro
has DNA instead of RNA primers
uses heat to unwind instead of helicase
only amplifies a segment of DNA
describe the growth curb of a cell culture in suspension
lag then log (exponential growth)
stationary
death due to toxic byproducts and running out of nutrients
describe the relationship between objective and eyepiece lens on a light microscope
objective forms a real intermediate image which is greatly magnified by the eye piece lens
40X objective lens with 10X eyepiece will magnify the object 400X
downside of electron microscope
object must be immobile and contained in a vacuum state
what is immunocytochemistry
uses labelled antibodies to target specific protein antigens in the cell via epitopes
what is flow cytometry
method to count and examine microscopic particle such as cells
suspend them in a stream of fluid and pass them through an electronic detection apparatus
explain gene knowndown
expression of organisms genes is reduced by genetic modification or treatment with a reagent
binding of a oligonucleotide can block transcription, degrade the mRNA transcript, or block mRNA translation
explain gene knockin
insertion of a protein coding cDNA sequence into a particular locus of an organisms chromosome resulting in enhanced expression
what is gene knockout and what is it used for
organism is engineered to carry genes that have been made inoperative
must take gene out of gamete or stem cell, If it hinders embryonic development can’t make it
used to learn the function of a gene that has been sequenced
what is restriction fragment length polymorphism
difference between two or more samples of homologous DNA molecules arising from differing locations of restriction sites
digested DNA is separated by length through gel electrophoresis and different cleavage sites can be found through normal genetic variation because everyone has different restriction sites and distances between these sites
what is single nucleotide polymorphism
normal genetic variation between individuals of the same species
explain the process of DNA extraction
cells are broken open to expose the DNA
membrane lipids are removed by adding a detergent
proteins are digested using a protease
DNA is precipitated from other cell components using alcohol
what is added to a bacterial DNA plasmid to make a clone
a gene for antibiotic resistance
a reporter gene for beta galactosidase/LacZ
known locations of restriction endonuclease sites
how do you make a recombinant plasmid
plasmid cut with restriction enzyme that only cleaves within the reporter gene
dna sequence to be cloned is also cut with the restriction enzyme
foreign dna inserted into the plasmid which doesn’t allow LacZ to regain function
after a recombinant plasmid is made how is it cloned
grown on an agar plate containing an antibiotic and Xgal
only bacteria that have obtained the new plasmid can grow because they have antibiotic resistance
because they have a non functional LacZ gene they appear white instead of blue (screening factor)
white are picked and grown in culture
how does DNA sequencing work
dideoxynucleotides are added to DNA
they lack the OH group required for phosphodiester bond formation resulting in chain termination
this results in DNA fragments of varying lengths
these undergo gel electrophoresis allowing the researcher to read the original sequence from the gel bottom to top in the 5’ to 3’ direction(template)
what is cDNA
DNA reverse transcribed from mRNA so it lacks introns normally found in eukaryotic DNA
if amplified in reverse transcription polymerase chain reaction its original gene was transcribed into RNA in vivo
what is in situ hybridization
uses a labelled complementary DNA or RNA probe to localize a specific DNA or RNA sequence in a portion of tissue to determine where a given gene is transcribed
what dose a northern blot tell us
identifies RNA fragments
allows one to observe a particular genes expression pattern
how can you distinguish southern northern and western blot
SNOW DROP
southern identify specific sequences of DNA
northern RNA
western protein
what is immunohistochemistry
used to determine protein expression
antibody binding to a known protein then a secondary antibody link to a fluorescent molecule recgonizes the primary antibody
similar to ISH but its proteins instead of dna/rna
what does X-ray crystallography tell us
method to determine the arrangement of atoms within a crystal
helps determine the 3D structure of proteins
