Part D: Lecture 32

Question 1

Electrophoresis (def.)

Answer 1

the separation of charged molecules using an electric field

Question 2

Gel material does what?

Answer 2

slows down migration of DNA

Question 3

2 types of gel material

Answer 3

agarose
acrylamide

Question 4

agarose separates pieces of ____

Answer 4

dsDNA > 100 bp

Question 5

acrylamide separates pieces of ____

Answer 5

dsDNA < 1 kb
ssDNA

Question 6

gel material preference

Answer 6

agarose gel (“cheap and edible”)

Question 7

acrylamide gel problems

Answer 7

expensive and poisonous

Question 8

Gel apparatus used

Answer 8

Slab gel in a buffer between negative and positive electrode

Question 9

buffer function in gel electrophoresis

Answer 9

conducts charge between electrode

Question 10

DNA mix for gel electrophoresis into well

Answer 10

DNA molecules + glycerol + loading/tracking dye

Question 11

glycerol function in DNA mix

Answer 11

DNA falls into well

Question 12

loading/tracking dye (dark blue) function

Answer 12

allows us to see wells are loaded

Question 13

shorter linear pieces of DNA moves _____

Answer 13

faster

Question 14

longer linear pieces of DNA moves _____

Answer 14

slower

Question 15

Band (def. gel electrophoresis)

Answer 15

where the DNA ends up

Question 16

Lane (def. gel electrophoresis)

Answer 16

the path that DNA move

Question 17

charge of loading/tracking dye

Answer 17

negative charge (same of DNA sample)

Question 18

visualizing DNA in gel electrophoresis

Answer 18

-Ethidium bromide
-RedSafe

Question 19

EtBR +DNA/RNA +UV = ___

Answer 19

orange fluorescence

Question 20

RedSafe +DNA/RNA +UV = ____

Answer 20

green fluorescence

Question 21

Molecular weight markers (def.)

Answer 21

dsDNA fragments of known sizes and amounts

Question 22

restriction map (def.)

Answer 22

map of where RE cuts on DNA

Question 23

restriction fragments (def.)

Answer 23

DNA molecules produced by restriction digest

Question 24

Order of bands and restriction fragments

Answer 24

order of bands ≠ order of restriction fragments

Question 25

The fastest bands are ____ (appearance) because the pieces are ___

Answer 25

fainter
shorter (less Etbr in DNA)

Question 26

Determining the length of DNA procedure

Answer 26

1. Run a gel with unknown DNA in one lane + MOL wt markers in another lane
2. Take a photo (black and white)
3. Measure distance each band travelled from well
   4.Sheet of semi-log graph paper (STRAIGHT LINE)
4. Plot distance of mol wt markers
5. Estimate size of unk band

Question 27

Gel purifying DNA function

Answer 27

an easy way to obtain specific DNA molecules from a diverse collection of DNA

Question 28

Gel purifying DNA method

Answer 28

1)run DNA in a agarose gel
2)cut out the band and separate the DNA from the agarose + remove agarose with gel extraction kit

Question 29

Confirming integrity of DNA procedure

Answer 29

1) genomic DNA isolation protocol
2)load on gel directly
2)digest with RE another load on gel

Question 30

speed that DNA > 20 kb runs ___

Answer 30

as a single band

Question 31

RNA contamination

Answer 31

smear at the bottom

Question 32

protein contamination

Answer 32

thick band at the top