Lecture 6 Flashcards
Gel separates dna based on what
Size and charge
Ideally charge to mass ratio is consistent —> only size
4 components for gel
- 2 electrodes
- buffer with ions
- porous gel
- evenly charged, linear polymer
Agarose or poly acrylamide bigger pores?
Agarose
Poly acrylamide is cross linked
Generally for gels, percentage of polymer rules
Low % = higher mw sample = bigger pores
High % = lower mw samples = smaller pores
What is the cross linking agent for poly acrylamide?
BIS acrylamide
Also needs per sulfate molecule
Which gel type is run vertically?
Poly acrylamide
= stacking gel on top of
Running gel on bottom
DNA charge
-2 per base pair
One neg per phosphate
Runs towards positive anode
Anode = pan out = p
Cathode = catheter = negative experience
For agarose gel, smaller molecules are closer to bottom or top of gel?
Bottom of gel
Smaller molecules travel further
Specific gel for protein
SDS-PAGE
SDS = detergent
PAGE = poly acrylamide gel electrophoresis
SDS function
Straightens out protein by forming micelles around hydrophobic protein components = in addition to heat, helps unfolding process
Mass to charge ratio with SDS
1 SDS / 2 amino acid
Why does RNA need to be treated with heat and formaldehyde?
RNA is linear and forms loops
Needs to be linearized
If 3 bands after plasma prep
Top band = nicked = not linear = not tight coils = slow
Middle band = linear dna = not super tight = sort of slow
Bottom band = supercoiled = runs fast
How to stain dna gels
Ethydium bromide
Positively charged = inserts itself between bases = conjugated aromatic rings = fluorescent under uv light
How to stain protein gels?
Coomassie blue
Dye binds to protein via positive charges and aromatic rings in protein
Or
Silver stains = very sensitive = agno3
Ag+ is reduced to metal silver
Western blot
Useful for identifying specific protein of interest
Transfer protein from gel to nitrocellulose membrane using electric field
Requires antibodies specific to protein of interest + secondary antibody that is labeled for detection
