Lecture 3.12

Lecture 3.12 - Fig. 12-1 Single-stranded sticky ends CH 3...

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Unformatted text preview: Fig. 12-1 Single-stranded sticky ends CH 3 CH 3 Table 12-1 Fig. 12-2 Fig. 12-3 Fig. 12-4 Fig. 12-5 Foreign DNA Sticky ends Vector Cloned DNA Introduction of recombinant vector into a host Add DNA ligase to form recombinant molecules Add vector cut with same restriction enzyme Cut with restriction enzyme Fig. 12-6 Ampicillin resistance pUC19 2686 base pairs Polylinker lacZ Order of restriction enzyme cut sites in polylinker Apo I - EcoR I Ban II - Sac I Acc65 I - Kpn I Ava I - BsoB I - Sma I - Xma I BamH I Xba I Acc I - Hinc II - Sal I BspM I - BfuA I Sbf I Pst I Sph I Hind III lacl Origin of DNA replication Fig. 12-7 AmpR lacZ Vector Foreign DNA Digestion with restriction enzyme Opened vector Join with DNA ligase Recyclized vector without insert Vector plus insert Transformants white ( -galactosidase inactive) Transformants blue ( -galactosidase active) Transform into Escherichia coli and select on ampicillin plates containing Xgal Fig. 12-8 Missing OH Dideoxy analog Direction of chain growth Normal deoxynucleotide DNA chain No free 3 -OH, replication will stop at this point Fig. 12-9 DNA strand to be sequenced Add DNA polymerase, mixture of all four deoxyribonucleotide triphosphates; separate into four reaction tubes A small amount of only one dideoxynucleotide triphosphate (ddGTP, ddATP, ddTTP, or ddCTP) added to each tube and reaction allowed to proceed Reaction products Radioactive DNA primer ddGTP ddATP ddTTP ddCTP G A T C 7 6 5 4 3 2 1 Reaction products separated by electrophoresis on gel and identified by autoradiography Sequence reads from bottom of gel as A G C T A A G. Sequence of unknown is 3 T C G A T T C 5 C A A A G G T Fig. 12-9aFig....
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Lecture 3.12 - Fig. 12-1 Single-stranded sticky ends CH 3...

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