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- can use restriction endonucleases to cut required gene from DNA (at specific base sequences) evenly (fforms blunt ends) or unevenly (leaving 1 strand with few more bases, these regions with unpaired/exposed bases called sticky ends)
- can instead isolate mRNA for the desired gene, and use reverse transcriptase to form single strand of cDNA (complimentary DNA), +ve easier to identify desired gene (as cell makes specific types of mRNA)
- plasmid cut at first marker gene (with same restriction endonucleases as to cut desired gene in DNA, so that sticky ends complimentary and DNA ligase forms phosphodiester bonds between fragments), first marker gene makes sure recombinant gene in plasmid (as it would have a function e.g flouresce, but when desired gene in place, stops function, so success if function stops), and second codes for antibiotic resistence (to see if bacteria have taken up plasmid by growing in antibiotic medium)
- plasmid with recombitant DNA transferred into host via transformation
- one method is to culture bacterial cells and plasmids in Ca rich sol and temp inc (bacterial membrane permeable now for plasmids to enter)
- another method is electroporation (small current applied to bacteria, so membranes more porous (now plasmids enter), control current power to prevent damage to membrane
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