gels & rd

Question 1

why do we use gels

Answer 1

way to measure length of DNA

Question 2

why do we need DNA length

Answer 2

determine if DNA is good enough to be sequenced

Question 3

what length makes DNA sequencable

Answer 3

greater than 300 base pairs

Question 4

properties of DNA

Answer 4

1) polar (soluble in water)
2) denatured & renatured
3) negatively charged
4) can be stained by Ethidium Bromide
5) reflects UV lights/rays

Question 5

agar function in agarose gel

Answer 5

general medium for gels to travel through

Question 6

buffer function in agarose gel

Answer 6

submerse gel and maintain charge distribution

Question 7

loading dye function in agarose gel

Answer 7

glycerol makes DNA denser than buffer and agar, see the the sample in each well

Question 8

ethidium bromide function in agarose gel

Answer 8

stain DNA

Question 9

components of agarose gel

Answer 9

agar, buffer, loading dye, ethidium bromide

Question 10

how to load gels

Answer 10

1) add loading dye to DNA sample
2) insert DNA into wells
3) insert a ladder
RUN THE GELS

Question 11

will smaller or larger DNA fragments move further?

Answer 11

smaller fragments will move faster because there is more space within the agar to move

Question 12

which end do you add DNA

Answer 12

negative end (cathode)

Question 13

why do we use RD

Answer 13

a way to double check our PCR (insert size)

Question 14

what is RD

Answer 14

uses restriction enzymes that cut DNA at specific points

Question 15

what restriction enzyme do we use

Answer 15

PVUII (pvu2)

Question 16

what are restriction enzymes

Answer 16

degrade foreign DNA in bacteria from viruses, cut at restriction sites

Question 17

if RE degrades foreign DNA, why doesn’t it degrade bacterial DNA

Answer 17

bacterial DNA has methyl on nitrogenous bases

Question 18

what site is our insert at

Answer 18

SfiiA and SfiiB sites

Question 19

why do we not cut at the insert

Answer 19

less expensive, cut too close to insert might cut into insert

Question 20

what do random cuts result in

Answer 20

smeared bands

Question 21

how to do mockup

Answer 21

1) set gel picture to b&w (see bands better)
2) label ladder (left) and cut/uncut lanes
3) determine length of insert

Question 22

what does the intensity/brightness of bands correspond to

Answer 22

size and number of fragments in DNA band

Question 23

what will happen if you leave the enzyme in the plasmid for a long time

Answer 23

more plasmid it will cut

Question 24

errors

Answer 24

1) partial digestion
2) contamination
3) lab error
4) puncture

Question 25

how many base pairs are needed in PCR

Answer 25

300 base pairs

Question 26

how many base pairs are needed in RDs

Answer 26

700 base pairs

Question 27

where is Ethidium Bromide found

Answer 27

in gel (Kathie & Lina thought they had cancer when they touched the loading dye but it’s in the gel)