1. Amgen Lab 2a & 4a

2. Lab 2a: Restriction Enzymes
Restriction enzymes are naturally found in Bacteria & Archaea .
They are thought to provide protection against invading viruses by hydrolyzing “foreign” DNA.
RE cut DNA at specific recognition nucleotide sequences called restriction sites
Called “molecular scissors”

3. Produces “sticky ends”

6. Engineering the Plasmid: ligation of rfp gene into p-ARA
Bruce Wallace
sticky end
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I

7. Recombinant plasmid of interest
pARA-R construct
Bruce Wallace
Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp
702bp
Hind III

8. Recombinant plasmid of interest
Restriction digest of pARA-R
Bruce Wallace
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
BamH I
Hind III

9. Restriction fragments after digest with Hind III and BamH I
Restriction analysis of pARA-R
Bruce Wallace
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
Hind III
BamH I
702bp

10. Prediction for restriction gel
Restriction analysis of pARA-R
Prediction for restriction gel M A+ A- M A+ A-
500
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3000
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5000
8000
10000

11. A- Lane: Nicked, Multimer, & “Regular” plasmid

12. Ladder A+ A-