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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
What tells you the sizes of DNA fragments in PCR run on a gel?
The ladder
26
When running a gel after PCR, things run towards the negative/positive charge?
Positive charge
27
When running a gel after PCR, light/heavy things go further?
Light
28
Why is it important to not leave the gel running for too long for analysis after PCR?
You may lose the lightest DNA fragments
29
Why do you need a buffer on the outside of the gel to run gel electrophoresis?
To help conduct electricity better
30
In mini-prep, what were each of the solutions (1-3) used for?
Solution 1 = Resuspension Solution 2 = Lysis Solution 3 = Neutralising
31
What was in the resuspension solution in the mini-prep?
Glucose Tris HCl EDTA
32
What was in the lysis solution in the mini-prep?
NaOH SDS
33
What was in the neutralising solution in the mini-prep?
Potassium Acetate Glacial acetic acid dH2O
34
What was glucose used for in the resuspension solution in the mini-prep?
Maintaining osmotic pressure, preventing the cells from bursting
35
What was Tris HCl used for in the resuspension solution in the mini-prep?
Maintaining pH
36
What was EDTA used for in the resuspension solution in the mini-prep?
Collating divalent cations preventing DNAse damaging plasmid Destabilises the cell wall
37
What was NaOH used for in the lysis solution in the mini-prep?
Disrupting H bonds between bases, converting dsDNA to ssDNA Destabilises the cell wall
38
What was SDS used for in the lysis solution in the mini-prep?
Denaturing proteins in the cell Solubilises the membrane
39
What was potassium acetate used for in the neutralising solution in the mini-prep?
Decreasing alkalinity H-bonds re-establish in plasmid DNA (But not genomic DNA - it can't do this)
40
What was glacial acetic acid used for in the neutralising solution in the mini-prep?
Maintaining pH
41
What was dH2O used for in the neutralising solution in the mini-prep?
Allowing dsDNA plasmid to dissolve, but other things can't dissolve so you needed to keep the liquid but not the pellet
42
Why was isopropanol used in the mini-prep?
It precipitates DNA, aggravating it and allowing it to fall out of solution
43
Why was ethanol used in the mini-prep?
To clean
44
For colony picking, why was LB broth used?
It provides nutrients allowing the bacteria to grow
45
For colony picking, why was kanamycin used?
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
Why was transfection carried out?
To introduce DNA into cells so mammalian cells can produce protein that can be harvested
47
Name some ways of transfection
Electroporation Calcium phosphate precipitation Lipid mediated lipofection Retroviral infection Microinjection
48
How does electroporation work?
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
Why was optimem used in transfection?
It keeps the cells stable Doesn't contain FBS which would inhibit across the membrane
50
Why were cells split 1:3 instead of 1:5 for transfection?
To make sure they were still in the growth phase to transfect better
51
Why was tris HCl used in cell lysis solution?
It prevents protein denaturation Maintains pH to inhibit proteases
52
Why was sucrose used in the cell lysis solution?
To maintain the osmotic pressure
53
Why was EDTA used in the cell lysis solution?
Collating agent Makes Mg and Ca unavailable Protease inhibitor
54
Why was EGTA used in the cell lysis solution?
Collating agent Makes Mg and Ca unavailable
55
Why was sodium fluoride in the cell lysis solution?
Protease inhibitor
56
Why was sodium pyrophosphate used in the cell lysis solution?
Protease inhibitor
57
Why was triton-X-100 used in the cell lysis solution?
It is a detergent, causes breakdown of membranes and gives access to proteins
58
Why was beta-mercaptoethanol used in cell lysis?
It breaks disulphide bonds and denatures proteins
59
Why was a protease inhibitor tablet used in cell lysis?
Stops protein degradation by proteases
60
Why was the cell lysis carried out over ice?
Preventing protein degradation by proteases
61
What is protein measured in?
Kiladaltons
62
What is DNA measured in?
Base pairs
63
Why was a Bradford assay carried out?
It caused a coulometric change to decipher the protein concentration by the generation of a standard curve
64
What does a Western Blot do?
Separated proteins via an SDS Page gel
65
Why was protein transferred to nitrocellulose in western blot?
To look at it transferred onto membrane to look for marker
66
Why was Ponceau S used in western blot?
To further visualise proteins. Ponceau S binds to all proteins allowing them to be visualised
67
Why was anti-GAPDH used in western blot?
It is present in all cells so used as positive control
68
Why was horseradish peroxidase used in western blot?
Produces light allowing the proteins to be seen on the film
69
Why was MOPS used in Western blot?
To run the gel and help conductivity
70
Why was 1x transfer buffer used in Western blot?
It washes Involved in conductivity of electric current through gel
71
Why was 1x TBST used in Western blot?
Acts as buffer Washes Tween helps things that are not unstuck come off
72
Why was dried milk used in Western blot?
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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