PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by:...

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

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Transcript of PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by:...

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

pKAN-R/pARA Sequence

Biotechnology Lab Program

Laboratory Protocols by:

Marty IkkandaPowerpoint by:

Anthony DauloPierce College, Woodland Hills, CA

V.1.2.4

Bruce Wallace

Page 2: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Restriction analysis of pKAN-R and pARA

pKAN-R

pARA

BamH I

Hind III

BamH IHind III

rfp702 bp

5408 bp

4058 bp

40 bp

Bruce Wallace

Page 3: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Restriction analysis of pKAN-R and pARA

BamH I Hind III

702bp

40 bp

BamH I Hind III

BamH IHind III

BamH IHind III

Restriction fragments after digest with Hind III and BamH I

4018 bp

4706 bp

Bruce Wallace

Page 4: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Ligation of pKAN-R/pARA restriction fragments

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

3’

5’

5’

3’

3’

3’

5’

5’5’

5’3’

3’

Bruce Wallace

Page 5: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Ligation of pKAN-R/pARA restriction fragments

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

Bruce Wallace

Page 6: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Preparing competent cells for transformation

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

Adhesion zone

Calcium ions

Bruce Wallace

Page 7: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Transforming Escherichia coli with pARA-R

Recombinant Plasmids

Competent Cells

pARA-R

Bruce Wallace

Page 8: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Calcium ions

pARA-R

Transforming Escherichia coli with pARA-R

Adhesion zone

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

Bruce Wallace

Page 9: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth

Bruce Wallace

Page 10: PKAN-R/pARA Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4.

Colony isolation and culture

Preparing an overnight culture of E. Coli for RFP expression

LB/amp/arabroth

Bruce Wallace


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