Restriction Enzymes and Ligation Flashcards
What is a vector?
a replicon designed to carry passenger DNA of interest.
What is a replicon?
piece of DNA that is able to function like a chromosome in vivo.
What is a plasmid?
a vector, small circular double stranded extrachromosomal DNA.
What does a plasmid consist of?
- replication origin
- an antibiotic resistance gene (amp)
- multiple cloning site
What is the mobility of double stranded plasmid on agarose gels?
top to bottom: nicked circular DNA, linear DNA, supercoiled DNA.
What did the experiment involve?
cut vectors PBR322 and PUC19 with Bam H1 and Pst1 separately and both together.
What are sticky ends?
one strand is longer than the other.
What are blunt ends?
both strands are of equal length.
What enzyme is the ligation reaction catalyzed by?
DNA ligase
What is the commonly used DNA ligase?
Bacteriophage T4
What is the role of T4 DNA ligase?
Plays an important role in linking together short pieces of phage during DNA replication.
Ligation Reaction
Joining linear DNA fragments together with covalent bonds. DNA ligase involves creating a phosphodiester bond between the 3’-hydroxyl group of one nucleotide and the 5’ phosphate group of another.
Which molecule is required as a cofactor for the ligation reaction?
ATP is required as a cofactor. ATP is hydrolyzed by DNA ligase which provides the energy for the reaction to move forward.
What ingredients does a ligation require?
- Two or more fragments of DNA.
- A buffer which contains ATP.
- T4 DNA ligase
Ligation on Gel
fewer fragments will appear on gel after ligation.
