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Mystery Plasmid B ??????

Mystery Plasmid B ??????. Preliminary Work. 18 Possible Plasmids 11 Available Enzymes Too much work… Simplify: 3 Digestions. First Set of Digestions. AseI, BamHI Double Digestion DNA 3 μ L (1000ng/ μ L) AseI .75 μ L BamHI .75 μ L BSA 1.5 μ L Buffer 3 1.5 μ L H 2 0 7.5 μ L

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Mystery Plasmid B ??????

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  1. Mystery Plasmid B ??????

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

  3. First Set of Digestions AseI, BamHI Double Digestion • DNA 3 μL (1000ng/μL) • AseI .75 μL • BamHI .75 μL • BSA 1.5 μL • Buffer 3 1.5 μL • H20 7.5 μL Total 15 μL AseI, NcoI Double Digestion • DNA 3 μL (1000ng/μL) • AseI .75 μL • NcoI1 .75 μL • BSA 1.5 μL • Buffer 3 1.5 μL • H20 7.5 μL Total 15 μL BglI Single Digestion • DNA 3 μL (1000ng/μL) • BglI 1 μL • Buffer 3 1.5 μL • H2O 9.5 μL Total 15 μL All Buffers 100% Efficiency

  4. 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 First Gel AseI, BamHI AseI, BamHI Uncut Plasmid BglI AseI, NcoI 2-Log Ladder 2-Log Ladder Uncut Plasmid BglI AseI, NcoI 2-Log Ladder 2-Log Ladder 10 kb 10 kb 4.0 kb 4.0 kb 3.0 kb 3.0 kb 2.0 kb 2.0 kb 1.5 kb 1.5 kb 1.2 kb 1.2 kb 1.0 kb 1.0 kb 0.5 kb 0.5 kb 0.1 kb 0.1 kb

  5. BglI Single Digestion Uncut Plasmid BglI 2-Log Ladder • Single Restriction Site • Linearizes DNA • Size of Plasmid: little greater than 4,000 bp ONLY PLASMIDS WITH 1 BglI Site pGLO 5371 bp pGPS3 4293 bp pACYC177 3941 bp pBAD-HisB 4092 bp pET17b 3306 bp Possible Couldn’t See Small Band pGPS1.1 4580 bp 234 bp 10 kb 4.0 kb 3.0 kb 2.0 kb 1.5 kb 1.2 kb 1.0 kb 0.5 kb 0.1 kb

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

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

  8. All Three Digestions

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

  10. Extra Digestions:Chose 5 additional digestions to confidently identify plasmid NdeI DNA 3 μL Buffer 2 1.5 μL NdeI 1 μL H20 9.5 μL Total 15 μL AseI, BglII DNA 3 μL Buffer 3 1.5 μL AseI .75 μL BglII .75 μL H20 7.5 μL Total 15 μL EcoRI, EcoRV DNA 3 μL Buffer 3 1.5 μL EcoRI .75 μL EcoRV .75 μL BSA 1.5 μL H20 7.5 μL Total 15 μL NdeI, SphI DNA 3 μL Buffer 2 1.5 μL NdeI .75 μL SphI .75 μL H20 9 μL Total 15 μL PstI DNA 3 μL Buffer 3 1.5 μL PstI 1 μL BSA 1.5 μL H20 8 μL Total 15 μL pBAD-HisB All Buffers 100% Efficiency

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

  12. My Mystery Plasmid is: pBAD-HisB

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