Amgen Lab 2a & 4a

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”

Produces “sticky ends”

Engineering the Plasmid: ligation of rfp gene into p-ARA

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

Bruce Wallace

pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

Bruce Wallace

Restriction digest of pARA-R

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

Bruce Wallace

Restriction analysis of pARA-R

BamH I Hind III

702bp

BamH IHind III

Restriction fragments after digest with Hind III and BamH I

4018 bp

Bruce Wallace

Restriction analysis of pARA-R

Prediction for restriction gel

M A+ A-

500

1000

1500

20003000400050008000

10000

M A+ A-

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

Ladder A+ A-