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•Verify recombination by electrophoresis.
•Digest of rfp gene. •Transform bacteria with recombinant plasmid.
•Recombination (ligation) of plasmid and rfp gene.
•Induce expression of rfp gene.•Observe bacteria. •Digest of plasmid
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Journal 12.02.11
1) Copy the steps on the other slide in the correct order.
2) Flip the pages in the book: which steps correspond to labs 2a, 4a, 5a?
3) At which step is there a process of gene expression?
(Can be answered in 3 columns)
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DO: Lab 2a – Restriction digest1. Mix reagents, as described in page 2a.3-Reaction buffer mix-Plasmid-Water (to one test tube)-Add enzyme: from teacher.
2. Place at 37oC for at least 1 hour.
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•PreLab 2a – Q & A• Animation: Plasmid digestion
Group papers: •Lab 1 – Conclusions page 1.6•Lab 2a – Conclusions page 2a.4
-Pour Gel (not in 2013)
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http://Plasmid Recombination
Gene Cloning Animation
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pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
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rfp – Gene for Red Fluorescent Protein, originally from the Sea Anemone Discosoma sp.
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To presenting students:The following slide is from the alternative lab sequence, where students also performed the ligation step.
The two plasmids: One served as the ‘source’ of the rfp gene, and one as the ‘vector’ which would turn into our pARA-R.
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Restriction analysis of pKAN-R and pARA
Bruce Wallace
BamHI
HindIII
BamHI
HindIII
pKAN-R5,512 bp
pARA4,872 bpPBAD-rfp
806 bp376 bp
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Restriction analysis of pKAN-R and pARA
Restriction fragments after digest with Hind III and BamH I
Bruce Wallace
4,706 bp
BamH I Hind III
806 bp
BamH IHind III
376 bp
BamH IHind III
BamH I Hind III
4,496 bp
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Engineering the Plasmid: ligation of rfp gene into p-ARA
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
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Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
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To presenting students:Refer to Lab3 (background and conclusions) in the student guide for more information.Key points: -Why 70oC incubation (Denaturation – what does this mean?)
- What does ligation have to do with ‘recombinant DNA’?