identifying an unknown plasmid avity norman cal poly san luis obispo
TRANSCRIPT
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