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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