8) Restriction Enzymes & 9) Vector Properties

what are restriction enzymes

enzymes that cut DNA at specific sites

blunt ends

produced by cutting phosphodiester backbone directly down the middle

why are blunt ends difficult to use after cutting

due. toa lack of overhang it’s difficult to splice into vector

steps to obtaining protein via cDNA

1) isolate mRNA from cell

2) use reverse transcriptase and synthesize single stranded cDNA using mRNA as template

3) convert ss cDNA into ds cDNA

4) add appropriate linkers / restriction enzyme sites to the ends of the ds cDNA

5) splice ds cDNA into vectors (plasmids)

6) place vectors (containing the small frgament) into host cells

features of vectors

cloning (insertion) site (multiple cloning site or MCS), OriC (replication site), “Marker” for antibiotic resistance (AmpR)

what are vectors

plasmids; small circular extra-chromosomal self-replicating pieces of DNA

vector additional elements

promoter (lac operon promoter) and lac I gene (repressor protein), purification tags (Histidine tags)

cDNA library

genes that have been actively transcribed