Electrophoresis

Question 1

How does electrophoresis work?

Answer 1

• DNA has negatively charged groups (PO4-)
• DNA will migrate in position to positive anode as opposite charges attract when electric field applied due to electric current
• detector records when DNA fragments pass detector window (camera paired with laser) and when it passes they become excited and fluoresce so can be picked up
• mass/charge ratio will determine how fast the DNA moves

Question 2

How do different length DNA move towards positive anode?

Answer 2

• 10mer and 100mer migrate towards positive anode at same speed
• mass/charge ratio is same for all lengths of DNA (number of base pairs) so all move at same speed

Question 3

How do we separate different lengths of DNA in gel electrophoresis?

Answer 3

• pass them through a selective medium: a gel (which is 80-99.9 % solvent by weight)
• gel has intertangled fibrils and trapped solvents
• this hinders the movement of larger molecules
• smaller more compacted DNA will move faster than rigid rod double helix
• separates by size and shape as mass/charge ratio is constant

Question 4

What do we get from electrophoresis and how do we interpret this?

Answer 4

• gels are typically shown with cathodic end where samples loaded at the top
• using a ladder lets you compare your sample with known lengths
• migration distance can be calibrated to make accurate estimates of intermediate lengths
• shorter strands move faster and therefore appear at bottom of gel

Question 5

What are the two types of gels used in electrophoresis?
- name
- structure
- casting method
- typical gel conc
- DNA size range

Answer 5

• agarose
• tangled fibrils (works like a physical gel)
• heat up to melt it then pour it to cool to cast into gel like structure
• gel conc is 0.5 - 2 %
• 50 - 30,000 base pair DNA size range
• poly(acrylamide) (PAGE)
• chemically crosslinked matrix (makes matrix and is more like a chemical gel)
• mix it and then radical polymerise it to make into gel
• 3.5 - 30 % gel conc
• 6 - 2000 base pair DNA size range

Question 6

What is most important thing that matters for gel electrophoresis?

Answer 6

• most important thing that matters is density of space of gel
• size exclusion and speed at which DNA can pass through it

Question 7

What will we pick gel based on?

Answer 7

• will pick based on size needed of DNA
• PAGE can cut off much smaller sizes of DNA than agarose as has more diffuse structure

Question 8

What is the difference in agarose and PAGE electrophoresis set up?

Answer 8

• agarose gel is flat
• gel in middle of electrodes
• slots (made using comb inside when cooling) to put sample in then fill up with buffer and apply potential
• PAGE gel is upright (+ anode at top and - cathode at bottom)
• fill up with buffer and apply potential

Question 9

What is loaded into gel other than the sample?

Answer 9

• dye for visualisation of loading
• dye for tracking electrophoresis
• glycerol, glucose or urea for weighing sample (help it sink into well)

Question 10

How does concentration of agarose gel affect number of base pairs/cut off?

What else has effect on cut off?

Answer 10

-as increase concentration, cut off decreases

• buffer and dye both have significant effect on cut off (dye is in there interacting with DNA so the size of dye and how it stops it flowing through has impact)

Question 11

Can we use UV light to visualise gel?

Answer 11

• DNA absorbs UV light so decreased transmission can be observed but UV light damages DNA within 15 seconds so NO

Question 12

What are two ways to visualise a gel? (which is better and why)

How do these methods work?

What is bad about them?

Answer 12

• fluorescent stain (ethidium bromide)
• irradiate with light so it fluoresces so can visualise
• visible stain - more approachable as do not have to irradiate it (has long term affect - decomposes sample over time as breaking down H bonds over time)
• these are intercalators (they bind to major groove of DNA)
• do not know how carcinogenic they are so must be careful when handling

Question 13

What is another way to visualise a gel?
- name
- how it works
- three properties
- cons of it

Answer 13

• 32P labelling
• exposure to photographic film
• t1/2 = 14 days
• beta emitter and mildly radioactive
• good but not brilliant due to smeared bands
• smeared bands due to isotropic variation and release of radiation (it is a beta emitter)

Question 14

Capillary electrophoresis
- explain structure
- structure for nucleic acids
- what can be used with
- what is obtained
- advantages
- disadvantages

Answer 14

• very similar to normal electrophoresis but has two different buffer solutions with fine capillary bridging between buffer solutions
• potential is applied from positive to negative
• electric field pushes DNA through capillary
• capillary filled with acrylamide gel to provide extra separation
• can be used with many types of chemical and biochemical samples
• get 1D graph (not 2D)
• time along bottom against intensity
• can’t pick out bands
• this is easier as more control and calibration than gel electrophoresis
• it is more sensitive to gel structure and conc of buffer than gel electrophoresis
• can’t pick out bands on graph
• can’t multiplex this in same way we can with gel electrophoresis where we can have lots of different wells