Amgen Lab 2a & 4a
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Transcript of Amgen Lab 2a & 4a
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
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20003000400050008000
10000
M A+ A-
A- Lane: Nicked, Multimer, & “Regular” plasmid
Ladder A+ A-