Lab 7-8 (UV & electrophoresis)

Question 1

DNA stain for electrophoresis (2)

Answer 1

1) Ethium bromide (EtBr) or

2) methylene blue

Question 2

Loading dyes purpose

Answer 2

To monitor the migration of DNA. So electrophoresis can be stopped before the DNA “falls off” the end of the gel.

Question 3

2 attributes of Ethium Bromide (EtBr)

Answer 3

1) it is very sensitive

2) it is carcinogenic

Question 4

How does Ethium Bromide (EtBr) work?

Answer 4

Ethium Bromide (EtBr) intercalates into the double helix and fluoresces brightly under UV light source

Question 5

Types of loading dyes (2) plus an aid to sink down DNA into the well

Answer 5

1) Bromophenol blue
2) Xylene cyanol
3) Glycerol: heavy compound to sink down DNA into the well

Question 6

Composition of semi-solid matrix

Answer 6

A gell composed of agarose or polyacrylamide.

Question 7

1) net Charge of DNA and acid or base

2) Movement of DNA

Answer 7

1) negative and weak acid

2) will move from the negative pole toward the positive pole

Question 8

What type of particles move faster and travel further in electrophoresis?

Answer 8

small particles

Question 9

What is applied to the gel and what produces?

Answer 9

an electric current is applied and an electric field is created.

Question 10

1) Charge of proteins in basic solution

2) Charge of proteins in acidic solution

Answer 10

1) negative charged

2) positive charge

Question 11

If a cell tries to replicate UV-damaged DNA with dimers present, it usually ________

Answer 11

dies

Question 12

Most UV damage to DNA is in the form of _________ __________

Answer 12

pyrimidine dimers (thymine and/or cytosine)

Question 13

How does Photoreactivation work?

Answer 13

a specific photoreactivating enzyme (photolyase) uses energy from visible light to SPLIT the covalent bond in the pyrimidine dimers.

Question 14

What organisms have photoreactivation?

Answer 14

Bacteria and yeast

Question 15

How is pyrimidine dimers repair done in humans?

Answer 15

“nucleotide excision repair”.

Endonuclease remove the dimers and DNA polymerase resynthesizes the DNA across that region.

Question 16

1) are the repair process very effective?

2) what type of source is needed to produce substantial killing and mutation in normal yeast cells

Answer 16

1) yes

2) a source of high energy UV-C (such as a germicidal lamp)

Question 17

Type of yeast we initially got in the lab

Answer 17

Red mutated yeast.

Question 18

What would be the result of the reverse mutation?

Answer 18

White colonies of yeast

Question 19

Yeast suspension used?

Answer 19

1 million cells per mL

Question 20

Survival curve

Answer 20

plotting the fraction of the irradiated cells that survive to form colonies against the time of UV exposure

Question 21

How do we get white colonies?

Answer 21

1) by reversion ( an exact reversal of the original mutation)
2) by suppression ( a second site mutation which returns the phenotype to normal)

Question 22

1) What gene in the yeast was mutant?

2) What type of colonies produced?

Answer 22

1) adenine - requiring mutant adn(-)

2) red colonies