1. Mystery Plasmid B ??????

2. Preliminary Work 18 Possible Plasmids 11 Available Enzymes Too much work… Simplify: 3 Digestions

3. First Set of Digestions BglI Single Digestion DNA3 μL (1000ng/μL) BglI1 μL Buffer 31.5 μL H 2 O9.5 μL Total15 μL AseI, BamHI Double Digestion DNA3 μL (1000ng/μL) AseI.75 μL BamHI.75 μL BSA1.5 μL Buffer 31.5 μL H 2 07.5 μL Total15 μL AseI, NcoI Double Digestion DNA3 μL (1000ng/μL) AseI.75 μL NcoI1.75 μL BSA1.5 μL Buffer 31.5 μL H 2 07.5 μL Total15 μL All Buffers 100% Efficiency

4. 2-Log Ladder Uncut PlasmidBglI AseI, BamHI AseI, NcoI 2-Log Ladder First Gel 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 2-Log Ladder Uncut PlasmidBglI AseI, BamHI AseI, NcoI 2-Log Ladder All complete digestions BglI: Single Restriction Site ~3,500 – 4,500 AseI, BamHI: 3 Restriction Sites ~3,500 - 2,500 ~1,500 – 1,000 ~200 – 100 AseI, NcoI: 2 Restriction Sites ~ 3,500 – 2,500 ~ 1,500 – 1,200

5. BglI Single Digestion Single Restriction Site Linearizes DNA Size of Plasmid: little greater than 4,000 bp ONLY PLASMIDS WITH 1 BglI Site pGLO5371 bp pGPS34293 bp pACYC1773941 bp pBAD-HisB4092 bp pET17b 3306 bp Possible Couldn’t See Small Band pGPS1.14580 bp 234 bp 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 2-Log Ladder Uncut PlasmidBglI

6. AseI, BamHI 3 Restriction Sites ~3,500 - 2,500 ~1,500 – 1,000 ~200 – 100 pBAD-HisB 2740 1180 172 pEcoli-HN-GFPuv 2998 1424 BglI AseI, BamHI AseI, NcoI 2-Log Ladder 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb BglI AseI, BamHI AseI, NcoI 2-Log Ladder pET11c 2922 1424 1142 125 59 pGEM-T-MC1-RFP 2437 1235 59

7. AseI, NcoI 2 Restriction Sites ~ 3,500 – 2,500 ~ 1,500 – 1,200 pACYC177 2183 1758 pBAD-HisB 2818 1274 BglI AseI, BamHI AseI, NcoI 2-Log Ladder 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb BglI AseI, NcoI 2-Log Ladder pGLO 3633 1738 pGPS3 2424 1869

8. All Three Digestions BglIAseI, BamHIAseI, NcoI pBAD-HisB pGLOpEcoli-HN-GFPuvpGLO pGPS3 pACYC177 pGPS1.1 pGEM-T-MC1-RFP pET17bpET11c

9. pBAD-HisB Gel labeled with calculated pBAD-HisB bands from restriction sites Match the gel perfectly! 2-Log Ladder Uncut PlasmidBglI AseI, BamHI AseI, NcoI 2-Log Ladder 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 2-Log Ladder Uncut PlasmidBglI AseI, BamHI AseI, NcoI 2-Log Ladder 2740 4092 1180 172 2818 1274

10. Extra Digestions: Chose 5 additional digestions to confidently identify plasmid EcoRI, EcoRV DNA3 μL Buffer 31.5 μL EcoRI.75 μL EcoRV.75 μL BSA1.5 μL H 2 07.5 μL Total15 μL NdeI DNA3 μL Buffer 21.5 μL NdeI1 μL H 2 09.5 μL Total15 μL AseI, BglII DNA3 μL Buffer 31.5 μL AseI.75 μL BglII.75 μL H 2 07.5 μL Total15 μL NdeI, SphI DNA3 μL Buffer 21.5 μL NdeI.75 μL SphI.75 μL H 2 09 μL Total15 μL PstI DNA3 μL Buffer 31.5 μL PstI 1 μL BSA1.5 μL H 2 08 μL Total15 μL All Buffers 100% Efficiency pBAD-HisB

11. 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 10 kb 3.0 kb 4.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb 2-Log Ladder Uncut Plasmid EcoRI, EcoRV NdeI AseI, BglII 2-Log Ladder NdeI, SphIPstI Final Digestion Expected pBAD-HisB Bands EcoRV, EcoRI 30301062 NdeI 2391 1701 AseI, BglII 29251167 NdeI, SphI 23911435266 Pst 4092 3030 1062 2391 1701 2925 1167 2391 1435 266 4092

12. My Mystery Plasmid is: pBAD-HisB