Vectors -the DNA carriers Carriers to allow replication of recombinant DNA (including the foreign DNA in the host cell. 1)Plasmids:extrachromosomalself-replicating DNA molecules Clal Origin of replication EcoRI Hindlll EcoRV Antibiotic-resistant genes BamHI Allow the host to grow on Sphl selective media Sall Pvul Can selectively amplify this Pstl- specific vector in the host cell Xmalll Nrul Multiple cloning sites Allow insertion of foreign DNA
Vectors - the DNA carriers Carriers to allow replication of recombinant DNA (including the foreign DNA in the host cell. 1) Plasmids: extrachromosomal self-replicating DNA molecules Origin of replication Antibiotic-resistant genes Allow the host to grow on selective media Can selectively amplify this specific vector in the host cell Multiple cloning sites Allow insertion of foreign DNA
Cloning a foreign DNA fragment into pBR322 Amp'Te pBR322 1.Cutting the vector and the foreign DNA with PstI respectively,to generate the sticky ends 2.combining the cut vector with the foreign DNA and incubate them with DNA ligase DNA ligase. 3.transforming E.coli with the DNA mixture.calcium salt solution,electroporation(电穿f孔). Figure 4.4 Tet
1.Cutting the vector and the foreign DNA with PstI respectively, to generate the sticky ends ． 2.combining the cut vector with the foreign DNA and incubate them with DNA ligase． 3.transforming E．coli with the DNA mixture．calcium salt solution ,electroporation(电穿孔)． Cloning a foreign DNA fragment into pBR322
DNA fragment Plasmid vector to be cloned How can the recombinant Enzymatically insert DNA into plasmid vector be sorted out? Recombinant plasmid A mixture Mix E.coli cells with (not only plasmids in presence of CaCl2 recombinant) Bacterial Culture on nutrient agar chromosome plates containing ampicillin Transformed Cells that do not Cell multiplication E.coli cell survives take up plasmid die on ampicillin plates Independent plasmid replication Colony of cells each containing copies of the same recombinant plasmid
How can the recombinant be sorted out? Figure 7-3 A mixture (not only recombinant)