Lab 5 Flashcards
Where do restriction enzymes cut?
At the DNA backbone.
Restriction enzyme site:
The target site from which the DNA of interest is attached to the plasmid.
Plasmid:
Extra-chromosomal DNA that replicates independently of a chromosome.
A recombinant plasmid:
A plasmid that contains genes from two different sources.
DNA ligase:
Glues two pieces of DNA together.
It uses Phosphodiester bonds to connect the backbones together and ATP is used as an energy source.
Another name for a plasmid:
Vector.
What does a vector not have:
An insert.
Most plasmids contain:
Antibiotic resistance gene.
Plasmids’ ORI:
Origin of replication.
This is needed for the plasmid to be replicated in the bacterium.
ORI is where replication begins.
Polylinker / Polycloning:
Contains several restriction enzyme sites that are very close together.
This is where the gene is inserted.
Bacteria Transformation:
The process of moving a plasmid into a bacteria.
pHUG21 contains:
A heme transport gene.
An ampicillin resistance gene.
Another name for restriction enzyme:
Restriction endonuclease.
Restriction endonuclease:
An enzyme that cuts the DNA at a specific DNA sequence within the DNA strands, rather than at the very end.
Types of ends a restriction enzyme can leave:
Sticky end [Cohesive/overhang]
Blunt end.
Another name for sticky end:
Cohesive end.
Overhang.
Palindromic sequence:
A sequence that reads the same backward and forwards.
Restriction enzymes cut at:
Specific sites.
Restriction enzymes recognize:
The entire sequence.
Blunt end:
No overhang, harder to hydrogen bond.
How many more pieces of inserts than plasmids do you need?
4 times as many.
Why do bacteria make restriction endonuclease:
Bacteria produce them to chop up DNA from a bacteriophage that may have infected the bacterium.
The plasmid and the DNA need to have the same:
Overhangs.
Methylase:
Methylate some of the bases in the sequence so the restriction enzyme cannot cut it.