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CVDM > Lab Skills > Flashcards

Lab Skills Flashcards

1
Q

Why was a Lowry assay carried out?

A

To determine a protein concentration in unknown solutions

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2
Q

The Lowry assay causes a colour change. How does this colour change occur?

A

You need the protein to interact with the solutions, and you need all reagents for the colour change to occur

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3
Q

Why was tissue culture carried out?

A

To passage cells, allowing the cells more space to grow, give them more nutrients, and keep them alive

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4
Q

Why will cells die if they aren’t passaged?

A

They release a toxin when they can’t grow anymore

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5
Q

What was contained in DMEM used for passaging cells?

A

Media
GlutaMAX
Foetal Bovine Serum
1% sodium pyruvate
1% pen/strep
Magnesium and calcium

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6
Q

What does GlutaMAX do to the cells?

A

It is an amino acid

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7
Q

What does foetal bovine serum do?

A

It provides nutrients and proteins and inhibits trypsin

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8
Q

Why was pen/strep in the DMEM solution?

A

To prevent contamination

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9
Q

What do Mg and Ca do?

A

Inhibits trypsin

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10
Q

Why was PBS used for the passaging of cells?

A

If you put water on the cells they will burst, and if you add anything with Mg or Ca it will inhibit trypsin

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11
Q

What does trypsin EDTA do?

A

Collates divalent cations

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12
Q

Why was the incubator set to 37˚C and 5% CO2?

A

This mimics the mammalian conditions under which cells grow, and the cells used were mammalian cells, ensuring that they have optimal growth

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13
Q

What colour does the media turn under acidic and alkaline conditions?

A

Acidic conditions - media turns yellow
Alkaline conditions - media turns pink

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14
Q

What does PCR do?

A

Amplifies segments of DNA

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15
Q

Name some uses of PCR

A

Crime scene evidence
Genotyping
Paternity testing
Cloning
Testing for viruses

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16
Q

What does dNTP do in PCR?

A

Helps build the new strand of DNA

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17
Q

What do primers do in PCR?

A

Help identify the sequence you are trying to amplify

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18
Q

What does MgCl2 do in PCR?

A

It is involved in buffering. Different concentrations are required depending on the primer used

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19
Q

What does the reaction buffer do in PCR?

A

It contains different salts and alongside MgCl2 helps the reaction occur

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20
Q

What does Taq Polymerase do in PCR?

A

It is an enzyme that catalyses the reaction

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21
Q

What are the steps of PCR?

A

Step 1 = denaturation. 95˚C for 1 minute, allows hydrogen bonds to break and DNA strands to separate
Step 2 = annealing. Lower temperature for 30 seconds to allow the primers to attach
Step 3 = Elongation and extension. 72˚C for 1 minute, allows Taq polymerase to make new strands of DNA
Repeated for 30-40 cycles

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22
Q

What stages in PCR may be variable?

A

The temperature for the annealing phase
The time for the elongation and extension phase

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23
Q

How many primers do you need for PCR and why?

A

At least 2 primers
Need to have a forward primer and a backwards primer

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24
Q

How did we determine whether there was WT or mutant DNA when we carried out PCR?

A

By putting the DNA on gel. The fragments are separated by size.
By looking at the weight of the product you could determine whether it was WT or mutant

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25
Q

What tells you the sizes of DNA fragments in PCR run on a gel?

A

The ladder

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26
Q

When running a gel after PCR, things run towards the negative/positive charge?

A

Positive charge

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27
Q

When running a gel after PCR, light/heavy things go further?

A

Light

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28
Q

Why is it important to not leave the gel running for too long for analysis after PCR?

A

You may lose the lightest DNA fragments

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29
Q

Why do you need a buffer on the outside of the gel to run gel electrophoresis?

A

To help conduct electricity better

30
Q

In mini-prep, what were each of the solutions (1-3) used for?

A

Solution 1 = Resuspension
Solution 2 = Lysis
Solution 3 = Neutralising

31
Q

What was in the resuspension solution in the mini-prep?

A

Glucose
Tris HCl
EDTA

32
Q

What was in the lysis solution in the mini-prep?

A

NaOH
SDS

33
Q

What was in the neutralising solution in the mini-prep?

A

Potassium Acetate
Glacial acetic acid
dH2O

34
Q

What was glucose used for in the resuspension solution in the mini-prep?

A

Maintaining osmotic pressure, preventing the cells from bursting

35
Q

What was Tris HCl used for in the resuspension solution in the mini-prep?

A

Maintaining pH

36
Q

What was EDTA used for in the resuspension solution in the mini-prep?

A

Collating divalent cations preventing DNAse damaging plasmid
Destabilises the cell wall

37
Q

What was NaOH used for in the lysis solution in the mini-prep?

A

Disrupting H bonds between bases, converting dsDNA to ssDNA
Destabilises the cell wall

38
Q

What was SDS used for in the lysis solution in the mini-prep?

A

Denaturing proteins in the cell
Solubilises the membrane

39
Q

What was potassium acetate used for in the neutralising solution in the mini-prep?

A

Decreasing alkalinity
H-bonds re-establish in plasmid DNA (But not genomic DNA - it can’t do this)

40
Q

What was glacial acetic acid used for in the neutralising solution in the mini-prep?

A

Maintaining pH

41
Q

What was dH2O used for in the neutralising solution in the mini-prep?

A

Allowing dsDNA plasmid to dissolve, but other things can’t dissolve so you needed to keep the liquid but not the pellet

42
Q

Why was isopropanol used in the mini-prep?

A

It precipitates DNA, aggravating it and allowing it to fall out of solution

43
Q

Why was ethanol used in the mini-prep?

A

To clean

44
Q

For colony picking, why was LB broth used?

A

It provides nutrients allowing the bacteria to grow

45
Q

For colony picking, why was kanamycin used?

A

The normal bacteria isn’t resistant to kanamycin but the bacteria we want to grow has a kanamycin resistant gene in it so is resistant, allowing other bacteria to be killed by kanamycin, leaving only the bacteria we want

46
Q

Why was transfection carried out?

A

To introduce DNA into cells so mammalian cells can produce protein that can be harvested

47
Q

Name some ways of transfection

A

Electroporation
Calcium phosphate precipitation
Lipid mediated lipofection
Retroviral infection
Microinjection

48
Q

How does electroporation work?

A

It causes the electrical potential across the cells to rise and lets negatively charged DNA in
It causes tiny holes in the membrane transiently

49
Q

Why was optimem used in transfection?

A

It keeps the cells stable
Doesn’t contain FBS which would inhibit across the membrane

50
Q

Why were cells split 1:3 instead of 1:5 for transfection?

A

To make sure they were still in the growth phase to transfect better

51
Q

Why was tris HCl used in cell lysis solution?

A

It prevents protein denaturation
Maintains pH to inhibit proteases

52
Q

Why was sucrose used in the cell lysis solution?

A

To maintain the osmotic pressure

53
Q

Why was EDTA used in the cell lysis solution?

A

Collating agent
Makes Mg and Ca unavailable
Protease inhibitor

54
Q

Why was EGTA used in the cell lysis solution?

A

Collating agent
Makes Mg and Ca unavailable

55
Q

Why was sodium fluoride in the cell lysis solution?

A

Protease inhibitor

56
Q

Why was sodium pyrophosphate used in the cell lysis solution?

A

Protease inhibitor

57
Q

Why was triton-X-100 used in the cell lysis solution?

A

It is a detergent, causes breakdown of membranes and gives access to proteins

58
Q

Why was beta-mercaptoethanol used in cell lysis?

A

It breaks disulphide bonds and denatures proteins

59
Q

Why was a protease inhibitor tablet used in cell lysis?

A

Stops protein degradation by proteases

60
Q

Why was the cell lysis carried out over ice?

A

Preventing protein degradation by proteases

61
Q

What is protein measured in?

A

Kiladaltons

62
Q

What is DNA measured in?

A

Base pairs

63
Q

Why was a Bradford assay carried out?

A

It caused a coulometric change to decipher the protein concentration by the generation of a standard curve

64
Q

What does a Western Blot do?

A

Separated proteins via an SDS Page gel

65
Q

Why was protein transferred to nitrocellulose in western blot?

A

To look at it transferred onto membrane to look for marker

66
Q

Why was Ponceau S used in western blot?

A

To further visualise proteins. Ponceau S binds to all proteins allowing them to be visualised

67
Q

Why was anti-GAPDH used in western blot?

A

It is present in all cells so used as positive control

68
Q

Why was horseradish peroxidase used in western blot?

A

Produces light allowing the proteins to be seen on the film

69
Q

Why was MOPS used in Western blot?

A

To run the gel and help conductivity

70
Q

Why was 1x transfer buffer used in Western blot?

A

It washes
Involved in conductivity of electric current through gel

71
Q

Why was 1x TBST used in Western blot?

A

Acts as buffer
Washes
Tween helps things that are not unstuck come off

72
Q

Why was dried milk used in Western blot?

A

It acts as a blocking agent - it contains proteins that gets rid of ‘noise’ from antibodies so you don’t have background coming up when trying to visualise the proteins

CVDM (16 decks)

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