1. pKAN-R/pARA Sequence Biotechnology Lab Program Marty Ikkanda
Bruce Wallace
pKAN-R/pARA Sequence
Laboratory Protocols by:
Marty Ikkanda
Powerpoint by:
Anthony Daulo
Pierce College, Woodland Hills, CA
V.1.2.4

2. pKAN-R pARA Restriction analysis of pKAN-R and pARA rfp Bruce Wallace
5408 bp
BamH I
rfp
702 bp
pARA
4058 bp
Hind III
BamH I
Hind III
40 bp

3. Restriction fragments after digest with Hind III and BamH I
Restriction analysis of pKAN-R and pARA
Bruce Wallace
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
BamH I
Hind III
4706 bp
Hind III
BamH I
702bp
Hind III
BamH I
40 bp

4. Ligation of pKAN-R/pARA restriction fragments
Bruce Wallace
sticky end
BamH I 3’ 5’ 3’
sticky end
Hind III 5’
sticky end
Hind III 5’ 3’
sticky end
BamH I 5’ 5’ 3’ 3’ 3’ 5’

5. Recombinant plasmid of interest
Ligation of pKAN-R/pARA restriction fragments
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Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp
702bp
Hind III

6. Preparing competent cells for transformation
Bruce Wallace
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions

7. Transforming Escherichia coli with pARA-R
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Competent Cells
pARA-R
Recombinant Plasmids

8. Transforming Escherichia coli with pARA-R
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Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
pARA-R

9. Growth of transformed bacteria on various plates
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P+ plates LB
LB/amp
LB/amp/ara
P- plates
No growth LB
LB/amp

10. Colony isolation and culture
Preparing an overnight culture of E. Coli for RFP expression
Bruce Wallace
Colony isolation and culture
LB/amp/ara
broth