Identifying an Unknown PlasmidAvity NormanCal Poly San Luis Obispo

Identifying an Unknown Plasmid

•Serial Cloner•PCR•Restriction mapping•Agarose gel electrophoresis

Possible plasmids:

PWOW

PG

EcoRI

SspI-HF

Q

PCHL

PG

EcoRI

EcoRI

SspI-HF

Q

PZAP

PG

EcoRI

EcoRI

SspI-HF

SspI-HF

Q

PSMRT

PQ

EcoRI

EcoRISspI-HF

G G

PNRD

P

Q

EcoRI

SspI-HFG

PCAT

PQ

EcoRI

SspI-HF EcoRI

SspI-HF

Serial Cloner

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

PCR

Image source: Wikimedia commons

PCR

PWOWPCHLPZAP

PG

Q

PSMRTPCATPNRD

PQ

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 MgCl2

• 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 MgCl2

• 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

Lane Contents Volume

12 Promega 1kb DNA ladder 5 µL

11 AB 1/10 dilution raw plasmid

8 µL

10 AB G-primer PCR product 5 µL

9 AB G-primer PCR product 8.8 µL

8 AB Q-primer PCR product 5 µL

7 AB Q-primer PCR product 7.8 µL

6 AN Q-primer PCR product 10 µL

5 AN Q-primer PCR product 4 µL

4 AN G-primer PCR product 10 µL

3 AN G-primer PCR product 4 µL

2 AN 1/10 dilution raw plasmid

7.6 µL

1 Promega 1kb DNA ladder 3.7 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

PQ

G PQ

G P

QPNRD

EcoRI

SSpI-HF

PSMRT EcoRI

EcoRISSpI-HF

PCAT EcoRI

SSpI-HF EcoRI

SSpI-HF

PSMRT PCAT PNRD

EcoRI 2 3127, 1033

2 3013, 931

1 3684

SSpI-HF 1 4610

2 2534, 1410

1 3684

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 4•0.5 µL SSpI-HF restriction enzyme

Incubate37°C83 minutes

Restriction mapping: ResultsLane Contents

Lane Contents Volume

12 Promega 1kb DNA ladder 5 µL

11 AS HindIII digest 10 µL

10 AS Cla digest 10 µL

9 AS raw plasmids ¼ dilution

7.8 µL

8 AB EcoRI digest 8.2 µL

7 AB raw plasmids ¼ dilution

9.4 µL

6 AB SSpI-HF digest 3.6 µL

5 blank -

4 AN raw plasmids ¼ dilution

10 µL

3 AN EcoRI digest 10 µL

2 AN SSpI-HF digest 10 µL

1 Promega 1kb DNA ladder 5 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.

PSMRT PCAT PNRD

EcoRI 2 3127, 1033

2 3013, 931

1 3684

SspI-HF

1 4610

2 2534, 1410

1 3684

Contents of AN samples

Lane Bands Size Identity

2 1 A: ~6500bp SspI-HF digested plasmid

3 2 B: ~5000bpC: ~1000bp

EcoRI digested fragments

4 1 D: ~5000 bp Undigested plasmidEcoRI 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

PQ

EcoRI

EcoRISSpI-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, 2013. 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