Biol-K 323: Gel Electrophoresis

Front 1

what are chromatography techniques used for?

Back 1

to separate one molecule from another

Front 2

chromatography techniques

Back 2

1. used to separate one molecule from another

2. different techniques used for different molecules

Front 3

types of chromatography

Back 3

1. protein chromatography

2. column chromatography (HPLC)

3. gas chromatography

4. thin layer/paper chromatography

Front 4

protein chromatography

Back 4

1. utilizes acrylamide gels and column chromatography

2. various matrices separate proteins based on charge, size or affinity for matrix

Front 5

HPLC

Back 5

1. high pressure liquid chromatography

2. type of column chromatography

3. done at higher pressure to speed up the process

Front 6

gas chromatography

Back 6

1. volatilizes liquids

2. separates the molecules as a gas

Front 7

thin layer/paper chromatography

Back 7

separates smaller molecules

Front 8

agarose gel electrophoresis

Back 8

1. separates DNA or RNA fragments

2. based on size of the fragment

Front 9

agarose

Back 9

1. derived from sea weed

2. polysaccharide

3. consists of long polymer comprised of alternating D-galactose molecules and a molecule similar to L-galactose

4. powder that will dissolve at near boiling temperatures

5. solidifies when agar cools below 40deg

6. long polymers create a matrix which contains pores between the linear strands

Front 10

pore size

Back 10

1. depends on the concentration of the agarose in the gel

2. vary from 50 nm to more than 200 nm depending on agar concentration

3. researcher controls size by varying the percent of agarose in gel

Front 11

higher precent of agarose

Back 11

the smaller the pores between strands

Front 12

casting a gel

Back 12

1. agarose is lightly boiled to dissolve the agarose

2. allowed to cool to about 60 degrees

4. agarose must cool significantly before pouring gel (may warp tray if gel is too hot)

5. combs (differ in size) placed in casting tray

6. pour gel

7. gel solidifies

8. creates wells for holding DNA sample

9.

Front 13

typical range of agarose in buffer?

Back 13

0.5% - 1.8%

Front 14

how does DNA migrate?

Back 14

1. Electric field causes DNA to migrate to positive pole

2. migrates through pores created by the agarose matrix

3. small pores migrate more quickly

Front 15

electric field

Back 15

1. allows DNA to migrate

2. DNA migrates to positive pole

Front 16

why does DNA move to positive pole?

Back 16

because DNA has a negatively charged phosphate backbone

Front 17

fragment size: which moves faster?

Back 17

1. small fragments of DNA move more easily and quickly

2. large fragments move much more slowly

Front 18

which gels are better for resolving high molecular weight fragments?

Back 18

low percentage gels

Front 19

which gels are better for resolving low molecular weight fragments?

Back 19

high percentage gels

Front 20

low percentage gels

Back 20

better for resolving high molecular weight fragments

Front 21

high percentage gels

Back 21

better for resolving low molecular weight fragments

Front 22

running a gel

Back 22

1. DNA samples placed in loading buffer

2. pipet in gel

3. run at a voltage b/w 30 - 120 V

Front 23

what is the loading buffer we use?

Back 23

10X stop buffer aka "blue juice"

Front 24

loading buffer

Back 24

1. 10X stop buffer ("blue juice")

2. loading buffer

3. used to prepare the gel for a run

4. contains glycerol (makes it heavier)

5. contains SDS (sharpens bands and gives better resolution)

6. contains bromophenol blue (adds color that matches same rate as a ~500 bp DNA fragment)

Front 25

blue juice

Back 25

loading buffer

10 X stop buffer

Front 26

why does loading buffer contain glycerol?

Back 26

1. to make DNA sample heavier than water

2. allows DNA to stay in well if properly pipetted

Front 27

why does loading buffer contain SDS?

Back 27

sharpens bands --> gives better resolution

Front 28

why is bromophenol blue contained in the loading buffer?

Back 28

1. adds a colored band

2. migrates on the gel at the same rate as a ~500 bp DNA fragment

Front 29

what does the loading buffer contain?

Back 29

1. glycerol

2. SDS

3. bromophenol blue

Front 30

running voltage

Back 30

1. b/w 30-120 V

2. higher voltage = runs quicker

3. slower voltage = better band quality

Front 31

pro and con of a higher voltage

Back 31

1. quick results

2. lower quality (for publication)

Front 32

pro and con of a lower voltage

Back 32

1. slow results

2. higher quality (for publication)

Front 33

gel staining

Back 33

1. gels must be stained to visualize bands of DNA

2. stains bind to DNA and fluoresce under UV light

3. EtBr is a common stain

4. other common stains: Sybr Green, Sybr gold, GelRed

Front 34

EtBr

Back 34

1. used to stain gels

2. allow bands to be detected under UV light

3. intercalating agent (mutagen)

Front 35

how does EtBr allow bands to be detected under UV light?

Back 35

EtBr intercalates into the DNA ladder --> fluoresce

Front 36

non mutagenic gel stains

Back 36

Sybr Green, Sybr gold, GelRed

Front 37

standards

Back 37

1. needed whenever you want to know the molecular weight of DNA fragments

2. fragments of a known base pair number

3. allows "calibration" of the gel

4. need one on every gel we run

5. common ladders: 100 b.p, HindiII digest of Lambda Phage DNA

Front 38

100 bp ladder

Back 38

fragments: 100bp, 200bp, 300 bp

Front 39

HindIII digest of Lambda Phage DNA

Back 39

1. Hind III digest of Lambda Phage DNA

2. most common standard

3. viral genome from Lambda Phage isolated

4. cut with restriction enzyme HindIII

5. yields DNA fragments of knowno molecular weights (base pairs)

Front 40

standard curve

Back 40

1. generated to allow you to determine the number of base pairs in any band of your gel

2. Log MW (bp) v distance migrated (cm/mm)

3. takes advantage of the fact the small fragments will migrate farther than larger ones

4. logarithmic relation bw size and distance migrated

5. must measure from the origin to center of each band in the standard lane

Front 41

knowing the MW of the standard allows us to what?

Back 41

plot the standard curve

Front 42

standard curve axises

Back 42

Log MW (bp) v Distance Migrated (cm/mm)

Front 43

origin of the well

Back 43

bottom of the well

Front 44

how to generate a standard cureve

Back 44

plot log MW of each band vs distance each band migrated

Front 45

produce a standard curve

Back 45

Front 46

what do we do after we generate a standard curve?

Back 46

measure how far the bands of the digest have migrated

Front 47

what can you do after you know the distance each band migrated on the gel?

Back 47

use the curve to determine the MW of each band

Front 48

how to determine the MW of each band after knowing the migratory distance?

Back 48

1. Find the distance on the X-axis

2. go up the graph until you make contact with the standard curve

3. find what the y-axis reads at that point of contact

4. take the inverse log of the Y value

5. you will now have the MW value of the band

Front 49

pictures****

Back 49

***

Front 50

what do we need to construct a restriction site map?

Back 50

base pair size of the ScaI, BglI and double digests DNA

Front 51

what can we do after knowing the base pair size of the DNA fragments cut by Sca1, Bgl1 and double digests?

Back 51

construct a restriction site map