Molecular - Cloning Flashcards
Traits of a good cloning vector
Multiple cloning sites; antibiotic resistance (Amp or Kan) or GFP, origin of replication
What are clones good for?
Looking at genomes - each plasmid will get one piece, and all the plasmids (10,000s) represent the genome
cDNA library
It is a snapshot of expression, made from mRNA
How do we know if a colony contains gene of interest?
blue/white screening: clone has ßgal gene, treat with Xgal and IPTG - when ßgal is active it cleaves Xgal and creates blue. If clone contains inserts, then ßgal doesn’t work and colony is white
What is important to consider with regards to the multiple cloning site?
Ensure that the rest of the plasmid has no cut sites!
What is Xgal?
A chlorinated disaccharide which is cleaved by ßgal and forms blue precipitate
Where would you put ßgal in cloning vector?
What the multiple cloning site to be in the middle of ßgal. this prevents its function when an insert is present and the colony will be white.
IPTG
Induces the ßgal gene in the plamid (triggers transcription of lac operon)
Role of alkaline phosphatase in cloning
Prevents plasmid ends from reannealing. It clips the terminal (P) on the ends so it doesn’t close up again. Only the insert can fall in there and close it up. The increases the number of white colonies.
Why would you include certain promoters in a plasmid?
Then the plasmid will be activated in tissues, ex. use actin promoter because it’s very common. Can also make it more specific.
Why would you include eukaryote and prokaryote promoters in a plasmid?
Makes it easier to transition between different systems, such as animal, invertebrate, and bacterial
