pBR322 as a Vector by Shannon Patrick https://padlet.com/spatri11/2piztkxf7xydwhul en-us 2022-02-04 19:04:41 UTC 2022-02-04 19:29:03 UTC hello@padlet.com 1st Step: Creating Recombinant DNA spatri11 https://padlet.com/spatri11/2piztkxf7xydwhul/wish/2030021828 1. Plasmid is cut with restriction enzyme that cleaves one time within one of the antibiotic resistance genes

2. pBR322 fragment is combined with target DNA molecule which has been cut with same enzyme

3. Mixture is treated with ligase to increase the probability of vector attaching to target DNA and not to itself
]]> 2022-02-04 19:13:23 UTC https://padlet.com/spatri11/2piztkxf7xydwhul/wish/2030021828 2nd Step: Uptake of Recombinant DNA by Bacteria spatri11 https://padlet.com/spatri11/2piztkxf7xydwhul/wish/2030024266 Heat Shock method:
1. E. coli in mid-log phase growth can be suspended in cold CaCl2

2. Thawed on ice in the presence of recombinant DNA, then heat shocked at 42oC for 2 min 

3. Followed by incubating on ice – enables recombinant DNA to enter the cytoplasm  


]]> 2022-02-04 19:14:57 UTC https://padlet.com/spatri11/2piztkxf7xydwhul/wish/2030024266 3rd Step: Selection spatri11 https://padlet.com/spatri11/2piztkxf7xydwhul/wish/2030026287 1. Bacteria are plated on Amp plates – cells that have intact pBR322 and recombinant pBR322 will survive 

2. Cells that grow on Amp-containing medium are transferred to Tet plates– the BamHI site where target DNA was added is within the Tetr gene, so transformed cells that have the recombinant DNA will be sensitive to Tet  

3. Replica plating done

3. Individual cultures of Tet-sensitive cells are established from each of the colonies on the Amp-agar plates
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