Identifying an Unknown Plasmid Avity Norman Cal Poly San Luis Obispo

Identifying an Unknown Plasmid Serial Cloner PCR Restriction mapping Agarose gel electrophoresis

Possible plasmids: PWOW P G EcoRI SspI-HF Q PCHL P G EcoRI SspI-HF Q PZAP P G EcoRI SspI-HF Q PSMRT P Q EcoRI SspI-HF G G PNR D P Q EcoRI SspI-HF G PCAT P Q EcoRI SspI-HF EcoRI SspI-HF

Serial Cloner Image source: serial-cloner.en.softonic.com

PCR Image source: Wikimedia commons

PCR PWOW PCHL PZAP P G Q PSMRT PCAT PNRD P Q G + P + G - P + Q + P + Q - P + G

PCR Master Mix G: 25.8 µL sterile water 12 µL 5X GoTaq Green buffer 2.4 µL 25mM MgCl 2 4 µL Primer G 4 µL Primer P 6 µL 10mM dNTP 1.8 µL GoTaq DNA polymerase Master Mix Q: 25.8 µL sterile water 12 µL 5X GoTaq Green buffer 2.4 µL 25mM MgCl 2 4 µL Primer Q 4 µL Primer P 6 µL 10mM dNTP 1.8 µL GoTaq DNA polymerase Plasmid dilution: 1 µL unknown plasmid 500 µL nanopure water PCR Mixture: 14 µL Master Mix G or Master Mix Q 1 µL plasmid dilution PCR cycles: 94.0°C 2:00 minutes 94.0°C 0:30 minutes 59.5°C 0:30 minutes 72.0°C 2.00 minutes 72.0°C 7.00 minutes 4.0°C hold 30 cycles Image source: eshop.eppendorf.ca

PCR: Results PCR products and raw plasmids run on a 1.0% agarose gel in 1X TAE buffer at 120V for 40 minutes. Lane Contents LaneContentsVolume 12Promega 1kb DNA ladder5 µL 11AB 1/10 dilution raw plasmid8 µL 10AB G-primer PCR product5 µL 9AB G-primer PCR product8.8 µL 8AB Q-primer PCR product5 µL 7AB Q-primer PCR product7.8 µL 6AN Q-primer PCR product10 µL 5AN Q-primer PCR product4 µL 4AN G-primer PCR product10 µL 3AN G-primer PCR product4 µL 2AN 1/10 dilution raw plasmid7.6 µL 1Promega 1kb DNA ladder3.7 µL Q G

PCR: Results PCR products and raw plasmids run on a 1.0% agarose gel in 1X TAE buffer at 120V for 40 minutes. Conclusion: PSMRT, PCAT, or PNRD Q G

Restriction mapping P Q G P Q G P Q PNR D EcoRI SSpI-HF PSMRT EcoRI SSpI-HF PCAT EcoRI SSpI-HF EcoRI SSpI-HF PSMRTPCATPNRD EcoRI2 3127, , SSpI-HF ,

Restriction mapping EcoRI digest: 2.5 µL unknown plasmid 6 µL sterile water 1 µL 10X EcoRI buffer 0.5 µL EcoRI restriction enzyme SSpI-HF digest: 2.5 µL unknown plasmid 6 µL sterile water 1 µL 10X NE Buffer µL SSpI-HF restriction enzyme Incubate 37°C 83 minutes

Restriction mapping: Results Lane Contents LaneContentsVolume 12Promega 1kb DNA ladder5 µL 11AS HindIII digest10 µL 10AS Cla digest10 µL 9AS raw plasmids ¼ dilution7.8 µL 8AB EcoRI digest8.2 µL 7AB raw plasmids ¼ dilution9.4 µL 6AB SSpI-HF digest3.6 µL 5blank- 4AN raw plasmids ¼ dilution10 µL 3AN EcoRI digest10 µL 2AN SSpI-HF digest10 µL 1Promega 1kb DNA ladder5 µL Digested and undigested plasmids run on a 1.0% agarose gel in 1X TAE buffer at 120V for 40 minutes. Lane 5 was intentionally left blank.

Restriction mapping: Results Digested and undigested plasmids run on a 1.0% agarose gel in 1X TAE buffer at 120V for 40 minutes. Lane 5 was intentionally left blank. PSMRTPCATPNRD EcoRI2 3127, , SspI-HF , Contents of AN samples LaneBandsSizeIdentity 21A: ~6500bpSspI-HF digested plasmid 32B: ~5000bp C: ~1000bp EcoRI digested fragments 41D: ~5000 bpUndigested plasmid EcoRI digest SspI-HF digest

Conclusion: PSMRT PCR ▫No product when primers P and G used ▫Product when primers P and Q used ▫Band size: 1700 base pairs Restriction mapping ▫2 bands from EcoRI digest ▫1 band from SspI-HF digest ▫Band sizes? PSMRT P Q EcoRI SSpI-HF G

Thank you! References: ▫Goers, John, Susan Elrod, Michael Black, and Ken Hillers. BIO/CHEM 475: Molecular Biology Laboratory Manual. San Luis Obispo: Cal Poly Readers, Print. Image sources: ▫serial-cloner.en.softonic.com ▫Wikimedia Commons ▫eshop.eppendorf.ca Special thanks to Dr. Sandra Clement and the Cal Poly Biological Sciences Department