Molecular Biology Flashcards
What conditions are necessary for bacterial growth ?
- Agar culture medium
- LB medium (nutrient rich content)
- Bunsen burner (decontamination)
What is a Plasmid ?
A plasmid is a double-stranded DNA molecule that is not from the chromosomes. Usually circular and non essential and found mainly in bacteria and yeast.
What is the “origin” Ori ?
It’s the DNA sequence at which the DNA replication is initiated.
What are restriction sites ?
Restriction sites are DNA sequences that allow the introduction or removal of DNA fragments.
What are MCS (multiple cloning site)
MCS are a concentration of multiple restriction sites in one region.
What are restriction enzymes ?
Restriction enzymes are enzymes that recognize and cleave the DNA at restriction sites.
What are promoters ?
Promoters are DNA sequences located upstream of a coding sequence. They are responsible for synthesising mRNA and subsequent proteins.
What are the main steps of plasmid cloning ?
Preparing the DNA sequence we want to insert : extraction from cells + digestion (cleaving using restriction enzymes) or Reverse transcription (cDNA) + PCR amplification.
Digesting the end of the target with RE to make it compatible with the plasmid.
Check the DNA target and isolate it (gel agarose migration + extraction).
What is the first step of plasmid cloning ?
Open the plasmid using a restriction enzyme (RE).
Then, introduce the DNA sequence of interest by ligation (using DNA ligase).
What is the second step of plasmid cloning ?
Plasmid validation : verify that our target gene has been inserted (enzymatic restriction + migration on agarose gel).
What is bacteria transformation in this context ?
It is the transformation of the bacteria in order to insert the plasmid of interest.
