AGE Flashcards
To avoid overheating, take note of the
Voltage
Ionic strength
Increased voltage…
Increased heat
Degradation of gel
Bad resolution
Increased ionic strength?
Increased conductivity
Increased heat
Migration pattern of DNA
Cathode to anode
TYPES OF ELECTROPHORESIS
AGE
PAGE
Most widely used
Larger range of separation
Low resolution
Agarose gel electrophoresis (AGE)
AGE
Range of Concentration:
0.5%-2%
AGE
Advantage:
Disadvantage:
It can separate large molecules like NA or DNA
Low resulting power
High resolution
Suitable for smaller molecules
Difficult to make and expensive
Polyacrylamide gel electrophoresis (PAGE)
PAGE
Range of Concentration:
3.5%-20%
PAGE
Advantage:
Disadvantage:
High Resolution
More cumbersome - difficult to make and it’s more expensive than AGE
Starch - medium
Cheapest
Poor resolution
Starch gel electrophoresis
Agarose vs Polyacrylamide
Horizontal or vertical?
Horizontal
Vertical
Agarose vs Polyacrylamide
Big gaps or small gaps in gel?
Bigger gaps that with
polyacrylamide
Smaller gaps
Agarose vs Polyacrylamide
Separates large molecules or small ones?
Both, but better for large molecules
Both, but better for small molecules
Agarose vs Polyacrylamide
Lets you tell small differences between sizes of bands?
No
Yes
Agarose vs Polyacrylamide
Type of molecules run in this type of gel
Mostly DNA
DNA or protein
The spaces in between the gel particles in the well are the_____.
It is where the sample will pass
pores
We put the sample in the____.
• Upon applying electric current, the DNA molecule will migrate slowly
well
Band
• Intact =
• Smudge =
good quality DNA
degraded DNA
Highly purified uncharged polysaccharide derived from agar
AGAROSE
Agarose
Dissolves when added to boiling liquid
When temperature lowers to about____, it gels/ solidifies
40°C
• The lower the concentration, the____ the pores
Good separation for…
• The higher the concentration, the____ the pores
Good separation for…
bigger; large DNA fragments
smaller; smaller DNA fragments
Held together by the formation of weak hydrogen and hydrophilic bonds (hydrocolloids)
Agarose gel
BUFFERS FOR ELECTROPHORESIS
Tris-Acetate EDTA (TAE)
Tris-Borate EDTA (TBE)
Common running buffer for agarose
Tris-Acetate EDTA (TAE)
TAE
Working solution:
Stock solution:
0.5 - 1.0 x
50 x
Stronger buffering capacity
Can be used for agarose or PAGE
Tris-Borate EDTA (TBE)
TBE
Working solution:
Stock solution:
0.25 x
5 x
TAE vs TBE
Difference in components
Glacial acetic acid
Boric acid
Intercalates DNA to its planar structure
ETHIDIUM BROMIDE (EtBr)
EtBr
UV absorbed by DNA at_____ is transmitted to the dye
160 nm
EtBr in UV light emits a ____color (fluorescence) at_____ detected by the naked eye
red-orange ; 590 nm
SAFER ALTERNATIVES TO ETHIDIUM BROMIDE (EtBr)
EtBr is carcinogenic and toxic
SYBR® Safe DNA Gel Stain
Gel Red®
is to determine the size of the molecule
Our reference on the estimation of the amount of base pairs our sample has or the size of our sample
Ladder
