104 Lecture 3, Gel Electrophoresis

Front 1

Gel purification

Back 1

- 4/11/06 lab
- Run gel (2% low melt - refers to not needing more than 60C to melt agarose)
2. Cut out DNA Fragments bands (3) - small, large, and control
3. Purify DNA Fragments from Gel - pour liquified DNA to silica which binds DNA when guanidine is present. TE (Tris-EDTA) (aka 10-1 for 10mM to 1mM) is a physiological pH buffer to elute DNA from resin
You can control DNA's solubility by controlling presence of ethanol/ isopropanol - the more there is, the lower the solubility of DNA.
4. Determine size of bands on the gel using the phi x 174 phage standard ladder.

Front 2

Agarose vs. Acrylamide

Back 2

Agarose is a sugar from seaweed, acrylamide is H2CCHCONH2
- Polyacrylamide is used for 5-500bp - it has a high resolving power (like differentiating between 24 and 25 bp)
- Agarose is used for 200-50,000bp - it has a high separation power(separating bands of different sizes)

Front 3

Agar vs. Agarose

Back 3

- both made from same material (extract from seaweeds)
- Agarose more pure - it doesn't have DNAses, charged species, etc). Agar doesn't need to be so pure.
- Low melting agarose is more pure whereas high melt agarose has different polysaccharide strands.

Front 4

DNA and EtBr Migration in electric field

Back 4

- DNA migrates to the red, positive, anode.
- EtBr (which is positively charged) migrates to the black, negatively charged cathode.
- the orientation of DNA in the gel is parallel to the direction of its motion (it moves vertically through the gel, slithering like a snake through the pores)

Front 5

Factors that affect rate of migration of DNA in agarose gels

Back 5

1. Size of DNA
2. Concentration of Agarose
3. Conformation of DNA
4. Voltage
5. Presence of EtBr
6. Ionic Strength of Buffer
7. DNA sample (what you load into the wells) composition

Front 6

Size

Back 6

- Rate of migration is inversely proportional to the number of base pairs
- On a semi log graph ( of the log of the number of base pairs (y-axis) vs. the distance migrated (linear, x axis), the curve starts out very negatively sloped, then less negatively sloped (p.42)

Front 7

Concentration of Agarose

Back 7

- As the concentration of the agarose increases, the drag force (friction) increases
- 2 % is used for 100-2000bp
- 1 % for 400 -6,000bp
- .5% for 1000-30,000bp
- plasmid size is out of range of separation for 2%, but may be seen on 1 and .5%.
- p.44 - as seen on the same semi log graph, as the concentration increases, the magnitude of the negative slope increases (meaning to travel the same distance, a band would have to be much shorter in a concentrated gel)

Front 8

Conformation of DNA

Back 8

- Supercoiled travels farthest
- when one strand of supercoiled is nicked, you get nicked or circular DNA, which travels the next farthest (its size in the gel is actually 1/2 its real size because it folds as it migrates through the gel - doesn't stay circular)
- the linear DNA fragments travel the least.

Front 9

Voltage

Back 9

- Rate of Migration is directly proportional to voltage
- you don't crank up voltage all the way up because 1. you get decreased resolution because the factor at which you multiply the rates for increased voltage is not linear
2. Melt the Gel from the heat (which is how increased resistance due to increased voltage manifests itself).

Front 10

Presence of EtBr

Back 10

- EtBr saturates and intercalates between DNA base pairs.
- It is by itself fluorescent, but the reason why we see DNA bands is not really because it is more concentrated where DNA is, or that it has run off the gel in the opposite direction, but because DNA-EtBr complex has higher fluorescence than EtBr by itself. This is because if DNA base pairs are aromatic, quench UV light and feed it to EtBr to increase its intensity of fluorescence. So if you have a lot of EtBr in the gel, you can still see the bands.
- DNA with EtBr travels slower than DNA without EtBR not really because of less mass or because EtBr wants to travel the opposite way but because intercalation increases rigidity of DNA (makes it a rod, rather than a wiggling snake that can go through the pores of the gel), increasing the friction or drag.

Front 11

Ionic Buffer Strength

Back 11

- Rate of migration of DNA is directly proportional to Ionic Strength of Buffer ions move current and DNA along the gel.
- .5X TBE is made up of 45mM Tris-Borate, pH 8, and 1mM EDTA.

Front 12

DNA Sample Composition

Back 12

- DNA
- 20mM TRIS (pH of 7.4 to keep DNA negatively charged, which is how it is at physiological pH)
- Bromophenol Blue (~300bp) - therefore, if you have 100bp fragments, you shouldn't let this dye front reach the bottom of the gel.
- Xylene cyanol (green, 4000bp) - bromphenol blue and this dye together give an idea of separation, and when you should stop the gel.
- Glycerol - increases density of the loading DNA sample composition so that it can be more dense than the buffer so that it can sink to the bottom of the well. Glycerol doesn't affect rate of migration
- Ethanol or Isopropanol - if DNA sample, after it is loaded doesn't go down to the bottom of the well, it is because there is too much ethanol or isopropanol, which make the sample less dense. Therefore, you add glycerol because it increases the density while not affecting migration.
- Salt - salt may or may not be present in the ladder standard and the DNA sample. But different salt concentrations result in different migrations (because of current carrying capacity) - therefore, salt presence can explain discrepancies between observed and expected size of DNA fragments.