Lab 7-8 (UV & electrophoresis) Flashcards
DNA stain for electrophoresis (2)
1) Ethium bromide (EtBr) or
2) methylene blue
Loading dyes purpose
To monitor the migration of DNA. So electrophoresis can be stopped before the DNA “falls off” the end of the gel.
2 attributes of Ethium Bromide (EtBr)
1) it is very sensitive
2) it is carcinogenic
How does Ethium Bromide (EtBr) work?
Ethium Bromide (EtBr) intercalates into the double helix and fluoresces brightly under UV light source
Types of loading dyes (2) plus an aid to sink down DNA into the well
1) Bromophenol blue
2) Xylene cyanol
3) Glycerol: heavy compound to sink down DNA into the well
Composition of semi-solid matrix
A gell composed of agarose or polyacrylamide.
1) net Charge of DNA and acid or base
2) Movement of DNA
1) negative and weak acid
2) will move from the negative pole toward the positive pole
What type of particles move faster and travel further in electrophoresis?
small particles
What is applied to the gel and what produces?
an electric current is applied and an electric field is created.
1) Charge of proteins in basic solution
2) Charge of proteins in acidic solution
1) negative charged
2) positive charge
If a cell tries to replicate UV-damaged DNA with dimers present, it usually ________
dies
Most UV damage to DNA is in the form of _________ __________
pyrimidine dimers (thymine and/or cytosine)
How does Photoreactivation work?
a specific photoreactivating enzyme (photolyase) uses energy from visible light to SPLIT the covalent bond in the pyrimidine dimers.
What organisms have photoreactivation?
Bacteria and yeast
How is pyrimidine dimers repair done in humans?
“nucleotide excision repair”.
Endonuclease remove the dimers and DNA polymerase resynthesizes the DNA across that region.
1) are the repair process very effective?
2) what type of source is needed to produce substantial killing and mutation in normal yeast cells
1) yes
2) a source of high energy UV-C (such as a germicidal lamp)
Type of yeast we initially got in the lab
Red mutated yeast.
What would be the result of the reverse mutation?
White colonies of yeast
Yeast suspension used?
1 million cells per mL
Survival curve
plotting the fraction of the irradiated cells that survive to form colonies against the time of UV exposure
How do we get white colonies?
1) by reversion ( an exact reversal of the original mutation)
2) by suppression ( a second site mutation which returns the phenotype to normal)
1) What gene in the yeast was mutant?
2) What type of colonies produced?
1) adenine - requiring mutant adn(-)
2) red colonies
