lab skills Flashcards
(42 cards)
what does a lowry do?
determine unknown concentrations of a protein
measures absorbance through colour change
what is BSA?
a protein used to generate a standard absorbance curve
why do we duplicate the experiment in the lowry?
to reduce errors (e.g. pipetting errors) and so we can spot if one result is very far off the curve
describe passaging
= 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
describe the contents of DMEM and what they do
FBS media (nutrients)
sodium pyruvate (energy boost)
penstrep (antibiotic)
why does DMEM have to be removed and washed with PBS before trypsin is added?
DMEM contains magnesium and calcium and FBS contains protease inhibitors which inhibit trypsin action.
what is PCR?
biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time
what are the environmental conditions for passaging?
37 degrees 5% C02
what are the stages of PCR?
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
what extra things are sued in PCR?
reaction buffer, DNTPS, magnesium chloride, primers (3 different), TAC enzyme
what happens after the PCR ?
it is run on a gel matrix. DNA is negative and has to push through towards the positive side - there is separation by size
what is the DNA ladder?
for sizing tells you how many base pairs
what is a plasmid?
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.
what are the 3 steps of purifying plasmid DNA from bacteria?
growth of bacterial culture
harvesting and lysis of the bacteria
purification of plasmid
describe alkaline lysis
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
describe the steps of preparation of plasmid DNA
E.coli pellet containing GFP plasmid + solution 1 (resuspension)
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E.coli suspension containing GFP plasmid + solution 2 (lysis)
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Alkaline lysis of DNA and protein + solution 3 (neutralisation)
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neutralisation causes precipitation of insoluble debris (protein chromosomal DNA)
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supernatant transfered to new tube
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alcohol precipitation (removes salts, small molecules)
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plasmid DNA (GFP) isolated
describe solution 1 of the alkaline lysis
FOR RESUSPENSION
glucose - maintains osmotic pressure
TRIS HCL - pH
EDTA - destabilises cell wall, primer for cell lysate, collates divalent cations, stops DNAase damaging plasmid
describe solution 2 of the alkaline lysis
FOR LYSIS
NaOH - disrupts H bonds between bases, converts to single stranded, breaks down cell wall
SDS - solubilises membrane, breakdown proteins of cells
describe solution 3 of the alkaline lysis
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
why are plasmids canamycin resistant?
so we can select for plasmids that have be successfully altered - stops everything growing
why is the supernatant cleaned with isopropanol?
to precipitate DNA. centrifuging causes DNA to gather at the bottom so debris can be removed.
clean with 70% ethanol - allows salt removal
why is a 1:3 split done before transfection?
for the right confluency to be achieved and to be in the correct growth stage this must be done 24 hours before
what is transfection?
= 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)
what level of confluency should cells be transfected at?
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
