Term
| what do we use to cut dna? |
|
Definition
| restriction endonucleases |
|
|
Term
| where do restriction endonucleuses come from? |
|
Definition
|
|
Term
| what were restriction endonucleuses originally used for? |
|
Definition
| cutting up bacteriophages |
|
|
Term
| how long are restriction sites? |
|
Definition
|
|
Term
| what are the different types of restriction endonucleases? |
|
Definition
I and III cut randomly
II recognize palidrome sequences |
|
|
Term
| what are the different types of ends that can occur from restriction? |
|
Definition
| 5' overhang, 3' overhang and blunt end |
|
|
Term
| how do you find the average length of a restriction fragment? |
|
Definition
| it is the probability of that particular sequence showing up |
|
|
Term
| how is the bacterial genome protected from its own restriction endonucleuses? |
|
Definition
| the enzyme methylase methylates bacterial genome |
|
|
Term
|
Definition
| the result of a bacterial genome (that is methylated) being replicated--the replication process results in two hemi-methylated strands |
|
|
Term
|
Definition
| fragments that can reanneal, like blunt ends. uses ligase to do so. |
|
|
Term
linkers
what do you do with them |
|
Definition
artificially synthesized dna strands with a restriction site on one end
add them to blunt end fragments with ligase.
then using a restriction endonulease we digest the ends making them have sticky ends so we can put things together |
|
|
Term
|
Definition
| dna molecule that is viable in a cell with and origin of replication and that can have dna cloned into them |
|
|
Term
| what are all the different types of vectors? |
|
Definition
plasmids
lambda phage
cosmids
Bacterial Artificial chromosome |
|
|
Term
|
Definition
| circular dna with origin of replication |
|
|
Term
|
Definition
| virus dna that infects bacterial cells |
|
|
Term
| how long is the lambda phage |
|
Definition
|
|
Term
| what is the basic construct of the lambda phage dna |
|
Definition
linear
the ends have 12 nuke sequences called cossites |
|
|
Term
| what is the construct of the cossite and what does it do |
|
Definition
| it is a 5' overhang but the sites are complimentary so the lambda dna can link easily |
|
|
Term
|
Definition
| when the cossites help pack lambda dna into the protein coat of the virus |
|
|
Term
|
Definition
| multiple lambda genome base paired at the cossites |
|
|
Term
| how do we use lambda phage |
|
Definition
-restriction endonuclease makes 2 cuts (left arm, right arm, internal fragment)
-foreign dna cut making fragments complimentary to the arms (frags go between arms)
-these recombinant frags ligate into concatamer
-add protein (coat)
-introduce to bacteria
-plaques form around a specific lambda phage |
|
|
Term
| what is the ideal length of a lambda phage? |
|
Definition
|
|
Term
| what is the origin of replication in plasmids? |
|
Definition
|
|
Term
|
Definition
Multiple Cloning Site in plasmids
where many different restriction endonucleases cut |
|
|
Term
| what is special about plasmids? |
|
Definition
| they have selectable markers |
|
|
Term
| what do selectable markers do? |
|
Definition
| tell you if you have sucessfully inserted foreign dna into the plasmid |
|
|
Term
| what are the two selectable markers for plasmids? |
|
Definition
ampr: plasmids should have the ampr gene making the bacteria cell they inhabit resistant to ampicillin. if a bacteria cell doesn't get its plasmid it will die on an ampicillin medium.
lacZ: most DNA inserts within a plasmid disrupt the lacZ gene. The lacZ gene makes b-galactose which cuts xgal and makes blue things. if a plasmid has a successful recombinant insert then it will be white |
|
|
Term
| how big of a plasmid can you get |
|
Definition
15kbp total
plasmid-3-5 kbp
insert=100bp-10kbp |
|
|
Term
|
Definition
| hybrid with a cossite in a plasmid |
|
|
Term
| what is the process of using a cosmid? |
|
Definition
-cut to create left and right arms
-insert foreign dna
-add protein coat (lambda phage)
-infect bacteria
-once in the cell the cossites ligate |
|
|
Term
| what is the ideal size of a cosmid? |
|
Definition
| 49 bp, you need that perfect amount to get it in the lambda proteins |
|
|
Term
| what is useful about the cosmid? |
|
Definition
it can replicate on its own but it doesnt make proteins so it is a non-functional virus that doesn't kill it's bacteria cell.
also has all the useful selectable markers |
|
|
Term
| what does the Bacteria Artificial Chromosome have? |
|
Definition
| ori, mcs, cam resistance, parA,. parB |
|
|
Term
| whats the big deal about BAC? |
|
Definition
| it can fit huge peices of recombinant dna into the vector. why? i don't have a fucking clue. |
|
|
Term
| what is the most amount of foreign dna that can fit inside a BAC? |
|
Definition
|
|
Term
|
Definition
| some magical thing in BAC that allows us to clone in massive amounts of recombinant dna |
|
|
