Required practicals 3 and 4 Flashcards
Potassium permanganate experiment, gel electrophoresis, acids and bases.
Calculate the molecular weight of KMnO4.
K = 39 g/mol
Mn = 55 g/mol
O = 16 g/mol
158g/mol
Calculate the number of moles required to make 250 ml of 100 mM KMnO4.
Give your answer in moles, to two significant figures.
0.025 mol
Now that you know how many moles of KMnO4 are required and you know the molecular mass of KMnO4 you should use your answers to the previous questions to calculate what mass of KMnO4 you require to make 250 ml of 100 mM solution.
3.95 g
How much 100 mM stock solution is required to make 100ml of 100 micromolar solution?
Give answer to 2.s.f. in ml.
0.10 ml
How do you dilute a concentrated acid?
Add the acid to the water.
When diluting a concentrated acid, what happens to the solution?
It will get hot.
When preparing a solution from NaOH pellets, what happens to the solution?
It will get hot.
What volume of concentrated (11.64 M) HCl is required to make 100 ml of 1 M HCl?
Give answer to one decimal place in ml.
8.6 ml
What should you do if you spill concentrated acid?
Rinse thoroughly with water.
What was the electrophoresis gel made of?
Agarose.
Why did we wear gloves to deal with KMnO4?
It is a powerful oxidising agent and will react quickly on skin. This will leave an insoluble brown stain of MnO2 which is not dangerous but will take a couple of days to fade.
Why is the weighing boat rinsed into the beaker?
To ensure all KMnO4 is transferred.
Why are the beaker and funnel also rinsed with distilled water?
To ensure complete transfer of MnO4.
How was the stock solution/dilutions of KMnO4 solution disposed of?
Stock = waste bottle.
Dilutions = down the sink with the tap running (to make waste as inert as possible).
Give the equation relating volume and concentration in terms of dilutions.
C1V1 = C2V2
(initial concentration x initial volume = new concentration x new volume)
What must be done before making/modifying corrections to the electrophoresis tank?
The power supply is switched off.
What must the buffer not spill over?
Leads and connections to the power supply.
Name the components of a gel electrophoresis tank.
Sample wells
Anode
Cathode
Power supply
Buffer solution
Agarose gel
Why is gel electrophoresis commonly used in molecular biology?
To separate DNA molecules, RNA molecules and protein molecules in complex mixtures according to their size and charge properties.
How are the sizes of the separated molecules estimated?
When molecular weight markers are run alongside samples.
When is agarose gel electrophoresis commonly used?
To separate DNA molecules.
How are the DNA molecules separated?
Negative DNA molecules pulled through agarose matrix within an electric field.
Shorter molecules move faster than longer ones.
List a property, other than the length of DNA molecules that is an important factor in migration rate.
The conformation (shape) of the DNA.
(Circular plasmids migrate the slowest and supercoiled move the fastest).
The voltage applied to the gel.
(Higher voltages give faster migration).
How are small DNA molecules separated?
Increasing the concentration of agarose gel so the migration speed is reduced.
Why must the voltage be limited?
It can heat up the gel and ultimately cause it to melt.
How is DNA viewed?
DNA is stained with a marker which allows it to be visualised under UV light.
Why must a centrifuge be balanced?
An unbalanced rotor can compromise the performance of your centrifuge, have an impact on the reliability of your testing results, and may even cause irreversible damage to your equipment.
Why was the gel completely submerged with Tris-Borate-EBTA (TBE) buffer?
The buffer provides ions that carry a current through the gel, and to maintain a constant pH.
How can you tell current is being produced?
Gas bubbles are visible from the cathode.
(Oxygen forms at anode and hydrogen at cathode).
Why did we wear gloves for gel electrophoresis?
Gels are usually stained with a DNA stain, which can be toxic.
Define cytotoxic (gel goes in cytotoxic waste bin).
Toxic to living cells.
Kills cells (including cancer cells).
Why is it important to calibrate the electrode of the pH meter?
Regular Calibrations Ensure Accurate pH measurements.
Comment on the accuracy of the acids and bases experiment.
Accurate:
- Regular calibrations of pH meter.
- Pellets of NaOH used instead of powder to
make 1 M and 0.1 M solution so all NaOH
transferred from weighing boat to
volumetric flask.
Not accurate:
- Only done once = results could be due to
chance.
- Smooth curve of best fit may not be the
best fit.
(Resulting in moles of HCl required to reach pH 7.40 being wrong and quoted pH range where HEPES solution acts as a buffer best).
Why does the pH curve have an initial gradual decrease rather than a sharp one?
The HEPES solution is resisting pH change.
Why is the curve flattest around pH 7-8?
HEPES molecules accept H+ from HCl.
After pH 8, why does the curve gradually decrease again?
All HEPES molecules have bound to H+, so as more HCl is added there is excess H+ and pH decreases.
