Mystery Plasmid B ??????. Preliminary Work 18 Possible Plasmids 11 Available Enzymes Too much work… Simplify: 3 Digestions.

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Presentation transcript:

Mystery Plasmid B ??????

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

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 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 μL Total15 μL All Buffers 100% Efficiency

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 ~1,500 – 1,000 ~200 – 100 AseI, NcoI: 2 Restriction Sites ~ 3,500 – 2,500 ~ 1,500 – 1,200

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 pACYC bp pBAD-HisB4092 bp pET17b 3306 bp Possible Couldn’t See Small Band pGPS 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

AseI, BamHI 3 Restriction Sites ~3, ,500 ~1,500 – 1,000 ~200 – 100 pBAD-HisB pEcoli-HN-GFPuv 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 pGEM-T-MC1-RFP

AseI, NcoI 2 Restriction Sites ~ 3,500 – 2,500 ~ 1,500 – 1,200 pACYC pBAD-HisB 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 pGPS

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

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

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 μL Total15 μL NdeI DNA3 μL Buffer 21.5 μL NdeI1 μL H μL Total15 μL AseI, BglII DNA3 μL Buffer 31.5 μL AseI.75 μL BglII.75 μL H 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

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 NdeI AseI, BglII NdeI, SphI Pst

My Mystery Plasmid is: pBAD-HisB