Common Lab Techniques

Question 1

describe how and SDS page works

Answer 1

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

Question 2

what is a western blot

Answer 2

use to detect specific PROTEINS in a sample based on ability to bind specific antibodies

Question 3

how is western blot performed

Answer 3

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

Question 4

gel electrophoresis separates dna molecules based on

Answer 4

size

Question 5

describe how gel electrophoresis works

Answer 5

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

Question 6

what are restriction enzymes and what do they produce

Answer 6

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

Question 7

what is a southern blot used for

Answer 7

detect specific DNA sequence

Question 8

how does southern plot work

Answer 8

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

Question 9

what does polymerase chain reaction do

Answer 9

amplify a single piece of DNA generating millions of copies of a particular DNA sequence

Question 10

what are the key reagents of PCR

Answer 10

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

Question 11

what are the 3 steps in the PCR process

Answer 11

1. denaturation: 90 degrees, DNA melts to single stranded molcules
2. annealing: 50 degrees, primers anneal upstream and downstream of the target sequence
3. elongation: 70 degrees, tan adds to template strand
   repeat 30 times

Question 12

4 differences from PCR and DNA replication

Answer 12

PCR in vitro
has DNA instead of RNA primers
uses heat to unwind instead of helicase
only amplifies a segment of DNA

Question 13

describe the growth curb of a cell culture in suspension

Answer 13

lag then log (exponential growth)
stationary
death due to toxic byproducts and running out of nutrients

Question 14

describe the relationship between objective and eyepiece lens on a light microscope

Answer 14

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

Question 15

downside of electron microscope

Answer 15

object must be immobile and contained in a vacuum state

Question 16

what is immunocytochemistry

Answer 16

uses labelled antibodies to target specific protein antigens in the cell via epitopes

Question 17

what is flow cytometry

Answer 17

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

Question 18

explain gene knowndown

Answer 18

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

Question 19

explain gene knockin

Answer 19

insertion of a protein coding cDNA sequence into a particular locus of an organisms chromosome resulting in enhanced expression

Question 20

what is gene knockout and what is it used for

Answer 20

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

Question 21

what is restriction fragment length polymorphism

Answer 21

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

Question 22

what is single nucleotide polymorphism

Answer 22

normal genetic variation between individuals of the same species

Question 23

explain the process of DNA extraction

Answer 23

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

Question 24

what is added to a bacterial DNA plasmid to make a clone

Answer 24

a gene for antibiotic resistance
a reporter gene for beta galactosidase/LacZ
known locations of restriction endonuclease sites

Question 25

how do you make a recombinant plasmid

Answer 25

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

Question 26

after a recombinant plasmid is made how is it cloned

Answer 26

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

Question 27

how does DNA sequencing work

Answer 27

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)

Question 28

what is cDNA

Answer 28

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

Question 29

what is in situ hybridization

Answer 29

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

Question 30

what dose a northern blot tell us

Answer 30

identifies RNA fragments

allows one to observe a particular genes expression pattern

Question 31

how can you distinguish southern northern and western blot

Answer 31

SNOW DROP
southern identify specific sequences of DNA
northern RNA
western protein

Question 32

what is immunohistochemistry

Answer 32

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

Question 33

what does X-ray crystallography tell us

Answer 33

method to determine the arrangement of atoms within a crystal
helps determine the 3D structure of proteins