RR4 Flashcards
What’s a plasmid?
Most common vectors: DNA molecules that replicate independently of chromosomes (mostly bacteria). Often round and double stranded.
What do restriction endonucleases do?
Cut dsDNA in a symmetrical fashion (creating sticky ends)
Describe recombinant DNA
Replication of DNA sequences that have been mixed with a vector through sticky ends complementarity. Allows for isolation, sequencing and manipulation of the purified fragment.
True or False? “Plasmids will only contain one restriction site”
False. You often need two just to cut the sequence you want. Careful not to cut that desired region.
What are the different regions of a plasmid?
ORI // Marker (resistance, fluorescence, etc.): tells if successful // Restriction enzymes
Describe recombinant DNA steps
Enzymatically insert DNA into plasmid // Add bacteria and plasmid to a bath // heat shock // Give nutrients in presence of Amp (or other antibiotics) // Only bacteria with plasmid survive // Each cell contains copies of recombinant plasmid
What are some uses for Recombinant DNA?
DNA libraries: for genes, for cDNA (from mRNA). Plasmid colonies contain a fragment each. // mRNA expression, co-regulation and localization // regulated expression of genes and protein production
How is cDNA made from mRNA?
A poly(A) tail is added to the mRNA // An oligo-dT primer is linked to the tail // reverse transcriptase synthesizes DNA // only the DNA is kept // It’s given a poly(G) tail // An oligo-dC primer is linked to the tail // dsDNA is obtained // After methylation (for protection), cDNA is attached to restriction site linkers // Cleave those sites to obtain sticky ends // Ligate to plasmid
True or False? “Eukaryotic genes can be mass replicated in bacteria through recombinant DNA”
True. Usually with lac promoter.
What’s the difference between transient and stable gene expression?
Transient: only stays in plasmid, lost after few generations.
Stable: Host chromosome is modified, won’t lose ability to produce new protein
