lab skills

Question 1

what does a lowry do?

Answer 1

determine unknown concentrations of a protein

measures absorbance through colour change

Question 2

what is BSA?

Answer 2

a protein used to generate a standard absorbance curve

Question 3

why do we duplicate the experiment in the lowry?

Answer 3

to reduce errors (e.g. pipetting errors) and so we can spot if one result is very far off the curve

Question 4

describe passaging

Answer 4

= the removal of the medium and transfer of a proportion of cells from a previous culture into fresh growth medium, a procedure that enables further propagation of the cell line or cell strain.

the growth of cells in culture proceeds from the lag phase following seeding to the log phase, where cells proliferate exponentially. when all teh available substrate is occupied and there is no room for expansion cell proliferation is greatly reduced or ceases entirely.

to keep them at optimal density for growth and proliferation we carry out passaging

Question 5

describe the contents of DMEM and what they do

Answer 5

FBS media (nutrients)
sodium pyruvate (energy boost)
penstrep (antibiotic)

Question 6

why does DMEM have to be removed and washed with PBS before trypsin is added?

Answer 6

DMEM contains magnesium and calcium and FBS contains protease inhibitors which inhibit trypsin action.

Question 7

what is PCR?

Answer 7

biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time

Question 8

what are the environmental conditions for passaging?

Answer 8

37 degrees 5% C02

Question 9

what are the stages of PCR?

Answer 9

1) denaturation - double stranded DNA templates are heated to seperate strands

2) annealing - primers bind to flanking regions of the target DNA

3) extension - DNA polymerase extends the 3’ end pf each primer along the template strands

these steps are repeated 25-35 times

Question 10

what extra things are sued in PCR?

Answer 10

reaction buffer, DNTPS, magnesium chloride, primers (3 different), TAC enzyme

Question 11

what happens after the PCR ?

Answer 11

it is run on a gel matrix. DNA is negative and has to push through towards the positive side - there is separation by size

Question 12

what is the DNA ladder?

Answer 12

for sizing tells you how many base pairs

Question 13

what is a plasmid?

Answer 13

double stranded, circular extra chromosomal DNA of a bacterium. it is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA.

Question 14

what are the 3 steps of purifying plasmid DNA from bacteria?

Answer 14

growth of bacterial culture
harvesting and lysis of the bacteria
purification of plasmid

Question 15

describe alkaline lysis

Answer 15

a method used to isolate plasmid DNA or other cell components by breaking cells open. bacteria is growth and lysed with an alkaline lysis buffer (consisting of SDS and a strong base sodium hydroxide). the detergent cleaves the phospholipid bilayer and the alkali denatures the proteins involved in structure.

through a series of steps involving agitation, precipitation, centrifugation and the removal of the supernatant, cellular debris is removed and the plasmid is isolated and purified

Question 16

describe the steps of preparation of plasmid DNA

Answer 16

E.coli pellet containing GFP plasmid + solution 1 (resuspension)
>
E.coli suspension containing GFP plasmid + solution 2 (lysis)
>
Alkaline lysis of DNA and protein + solution 3 (neutralisation)
>
neutralisation causes precipitation of insoluble debris (protein chromosomal DNA)
>
supernatant transfered to new tube
>
alcohol precipitation (removes salts, small molecules)
>
plasmid DNA (GFP) isolated

Question 17

describe solution 1 of the alkaline lysis

Answer 17

FOR RESUSPENSION

glucose - maintains osmotic pressure

TRIS HCL - pH

EDTA - destabilises cell wall, primer for cell lysate, collates divalent cations, stops DNAase damaging plasmid

Question 18

describe solution 2 of the alkaline lysis

Answer 18

FOR LYSIS

NaOH - disrupts H bonds between bases, converts to single stranded, breaks down cell wall

SDS - solubilises membrane, breakdown proteins of cells

Question 19

describe solution 3 of the alkaline lysis

Answer 19

FOR NEUTRALISATION

potassium acetate - decreases alkalinity so H bonds can re-establish and renature DNA

glacial acetic acid - for pH and so double stranded DNA can dissolve

Question 20

why are plasmids canamycin resistant?

Answer 20

so we can select for plasmids that have be successfully altered - stops everything growing

Question 21

why is the supernatant cleaned with isopropanol?

Answer 21

to precipitate DNA. centrifuging causes DNA to gather at the bottom so debris can be removed.

clean with 70% ethanol - allows salt removal

Question 22

why is a 1:3 split done before transfection?

Answer 22

for the right confluency to be achieved and to be in the correct growth stage this must be done 24 hours before

Question 23

what is transfection?

Answer 23

= introduction of foreign DNA into the nucleus of eukaryotic cells to then get the DNA to produce a protein.

stable transfections have integrated foreign DNA in their genome

transient transfections - foreign NDA does not integrate in the genome but genes are expressed for a limited time (24-96 hours)

Question 24

what level of confluency should cells be transfected at?

Answer 24

40-80% confluency. too few cells can cause cell cultures to grow properly without cell-to-cell contact. too many cells result in contact inhibition, making cells resistant to uptake of DNA and other macromolecules

Question 25

what are ways in which transfections can be done?

Answer 25

calcium phosphate precipitation
electroporation
lipofection
retroviral infection

Question 26

what is electroporation?

Answer 26

opens pores in cell by changing the electrical potential of the cell membrane.

Question 27

what are advantages and disadvantages of electroporation?

Answer 27

advantages - nonchemical method doesn’t alter biological structure or function of cells, easy to perform, high efficiency, can be used on lots of cell types

disadvantages - cell mortality is using suboptimal conditions.

Question 28

what does the lysis buffer do in harvesting?

Answer 28

lyse protein
protect the protein by making sure nothing will destroy it

Question 29

what are the other aspects of havesting?

Answer 29

TRIS HCL - prevents denaturation
sodium fluoride/ pyrophosphide - protease inhibitor
b- mercaproethanol - causes a linear protein shape

use on ice as the protease inhibitor works better on ice (stops denaturation)

Question 30

what is a bradford assay

Answer 30

another way of working our concentration of protein by absorbance

it is more modern and popular than lowry (lowry used for specific experiments)

Question 31

what is a western blot?

Answer 31

used to detect protein by size and determine quantity

used to study drug efficiency/ tx, genetic manipulation, protein interactions etc

Question 32

why cant you touch the nitrocellulose in western blot?

Answer 32

protein will transfer

Question 33

what does the PAGE gel used in western blot contain?

Answer 33

polyacrylamide

Question 34

what is the function of MOPS or other transfer buffers?

Answer 34

to allow electrical current to flow

Question 35

what does milk do in WB?

Answer 35

blocking agent stops unspecific binding of antibodiess

Question 36

what is ponceau S?

Answer 36

sticks to protein to show that the transfer works

Question 37

how does the electrical current work in WB?

Answer 37

goes up the gel then through the gel on to the nitrocellulose

Question 38

what is GADPHs role in the WB?

Answer 38

positive control

Question 39

what does horse radish peroxidase do in WB?

Answer 39

reacts with ECL to produce light which is then picked up by photographic film.

Question 40

what antibodies are used in western blot?

Answer 40

primary antibody (either anti-GFP or anti-GADPH) and then secondary antibody (anti-rabbit)

secondary antibody reacts with horse radish peroxidase (then this reacts with ECL)

Question 41

what is the moles calculation?

Answer 41

mol = grams x 1/MM

Question 42

what is the molarity calculation?

Answer 42

M = mol/ volume