•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 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
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’?