•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

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)

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.

•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)

http://Plasmid Recombination

Gene Cloning Animation

pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

rfp – Gene for Red Fluorescent Protein, originally from the Sea Anemone Discosoma sp.

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.

Restriction analysis of pKAN-R and pARA

Bruce Wallace

BamHI

HindIII

BamHI

HindIII

pKAN-R5,512 bp

pARA4,872 bpPBAD-rfp

806 bp376 bp

Restriction analysis of pKAN-R and pARA

Restriction fragments after digest with Hind III and BamH IBruce 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

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

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

Restriction digest of pARA-R

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

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’?