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.
Restriction enzyme modification:
Made up of the restriction enzyme and the methylase.
Why is modification done:
To prevent degradation of the host DNA by its restriction endonuclease.
Where is methylation done:
On the bases.
Restriction endonuclease nomenclature
Ex: E. coli RY13
1.) The first letter represents:
2.) The 2nd and 3rd letters represents:
3.) The second capital letter represents:
4.) The number represents:
EcoR1
1.) The genus.
2.) Species the enzyme was isolated.
3.) The strain.
4.) The order in which the enzyme was
discovered.
How do molecular biologists use restriction enzymes:
They use them to cut up DNA of interest into smaller pieces which are easier to manipulate.
Restriction enzyme cuts:
On both sides of the gene of interest.
kB stands for:
Kilobases.
Recombinant plasmids can be used by researchers to:
Overexpress a gene so protein can be purified and studied more closely.
Expression vectors:
Vectors that are used to overexpress a gene.
Restriction enzyme map can:
Help you create a plasmid that contains only a certain gene.
