Exam 1- Lecture 4 Flashcards
What acid is used to separate DNA and RNA
Phenol
How is phenol used to separate DNA and proteins
phenol is mixed with aqueous sample of DNA and proteins
proteins dissolve in phenol layer
nucleic acids form aqueous layer
layers separated through centrifugation
After using phenol to separate DNA from proteins, what enzyme is used to digest RNA into ribonucleotides
RNase
To separate DNA from ribonucleotides, alcohol is added and
DNA falls out of aqueous phase and is isolated
ribonucleotides stay soluble in solution
What is used to separate DNA fragments by size
gel electrophoresis
How does gel electrophoresis separate DNA fragments by size
electric current is passed through gel, negatively charged fragments move away from negative electrode
Do shorter DNA molecules move farther or closer to
they move farther than longer molecules
What are used to cut bacterial enzymes at specific recognition sites on DNA
restriction enzymes/endonucleases
What type of DNA can restriction enzymes not cut
their own DNA because it is methylated
What type of restriction enzyme is most useful for genetic engineering
Type 2
Where do type 2 restriction enzymes cut DNA
in the middle of recognition sequence
What is the function of DNA ligase
to link or ligate two different DNA fragments that have been cut using restriction enzymes
How could you make ligase more efficient
By having the two DNA fragments have the same overhanging sticky ends
In order to detect specific DNA molecules radioactively what is inserted into the molecules during replication
phosphate group
phosphorothioate group
What does autoradiography identify radioactive DNA as
“hot”
What will radioactive DNA look like during autoradiography?
It will turn gel surrounding it black
How is fluorescence used to determine nucleic acids
fluorescently tagged nucleotides incorporated into 3’ end of DNA
beam of light excites fluorescent tag
releases light
What is a Southern blot used for
form hybrid DNA molecule to determine if sample DNA has homologous sequence to another DNA molecule
What are Northern blots used for?
determine if sample of mRNA has homologous sequence to DNA probe
Why are Northern blots preferred to use for larger genomes
Because all of the introns are removed
What is the FISH technique
labeled probe is incubated with cells that have had their DNA denatured by heat
probe hybridizes to its homologous sequence on chromosome
What are cloning vectors
specialized plasmids that will hold any piece of foreign DNA for further study or manipulation
What is the polylinker region
region in cloning vector with multiple restriction enzyme sites
What are two ways inserts are detected within plasmids
resistance to antibiotic
synthesis of B-galactosidase
