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Transposon-mediated Mutagenesis
Step 1 - Amplify ORF from MG1655
1.0 ul genomic DNA (30ng/ul)
5.0 ul gene specific primer mix
4.0 ul 2.5 mM dNTP
5.0 ul 10x Turbo Pfu buffer
0.7 ul Turbo Pfu
34.3 ul sterile distilled H2O
50 ul
95°C 95°C 55°C 72°C 72°C 4°C
1min 15sec 15sec 4min 5min forever
25 cycles
Step 2 - Transposition reaction
6 ul ORF PCR product (~100-200ng)
2 ul Tn (Transposon, 200ng = 0.2pM)
1 ul 10x Tn reaction buffer (Epicentre)
1 ul Tnase (Transposase; Epicentre)
10 ul
Vortex, spin down
37°C for 2hr
Add 1 ul stop solution, 10min 70°C
Purify by passing through G50 column
Step 3 – Transformation
MG1655 cells harboring the pKD46 plasmid (Datsenko and Wanner, 2000, PNAS 97(12):6640-5) are induced with arabinose to activate expression of the lRed genes and prepared for electroporation.
1. Mix following on ice:
4.4 ul transposition reaction
40 ul MG1655 w/pKD46 electrocompetent cells
2. Transfer entire volume (~45ul) to chilled cubette (0.1cm gap), kept on ice
3. Electroporate with the following settings (for BioRad Pulse Controller)
Low Range 200
High Range 500
Capacitance (uF): -25
Total Volts: 1.8kV
4. Resuspend cells with 1ml LB and transfer contents to a 48-well growth block
5. Outgrow at 37°C for 1hr
6. Spread entire outgrowth on a corresponding labeled LB + Amp(100ug/ml)/Kan (50ug/ml) plate in a hood and air dry.
7. Grow at 300 (to maintain pKD46). (1- 2 days)
Step 4- Picking
Day 1
1. Streak 2 colonies to single colonies on LB + Amp(100ug/ml)/Kan (50ug/ml) plates. These will be the first colonies to verify.
2. Pick 3 additional colonies into separate 96-well flat bottom plates containing 200ul Freezing media + Amp(100ug/ml)/Kan(50ug/ml) maintaining original well location.
3. Grow 300 overnight.
Day 2:
1. Grow 1 colony from each streak plate in a 96-well block with each well containing 1ml Freezing media + Amp(100mg/ml)/Kan(50mg/ml). Grow 300 overnight.
2. Freeze overnights of the other 3 plates.
Step 5– Verify mutation by Culture PCR
1. Prepare sample
Mix culture thoroughly.
Transfer 20ul of culture into a 96-well plate and dilute with 80ul H2O Mix thoroughly
2. PCR Reaction.
5 ul diluted culture
4 ul gene specific primer mix (same as in Step 1)
2.5 ul ExTaq premix
16 ul H2O
50 ul
95°C 94°C 55°C 72°C 72°C 4°C
5min 30sec 15 sec 4min 5min forever
30cycles
3. Run 7 ul on 1 % test gel in 0.5x TAE to check.
4. Analyze gel results; mutants will be ~1.2kb larger than original gene length.
Step 6 - Confirm mutations by sequencing
1. EXOSAP clean up
- Transfer 10ul of PCR product for all genes with mutant sized fragments into a separate PCR plate
- Add 4ul ExoSAP-IT (USB) to each reaction
- Spin briefly
- React @ 37°C for 30min, then 15min@ 80°C
2. Sequence
1. Add a mix of the following to each ExoSAP reaction:
1ul of primer (KAN-2 FP-1 @ 10uM -Epicentre)
2ul Big Dye dilution Buffer (Promega)
3ul Big Dye2. Reaction conditions: (10sec@96C; 5sec@50C; 4min@60C) for 25cycles.
3. Purify through a G50 column
4. Dry plate and run on sequencer
3. Make tube stocks of confirmed mutants
Step 7 - Curing the temperature sensitive pKD46 plasmid
1. Streak confirmed mutants on a LB + Kan plate (from the above stock). Grow at 43° overnight (non-permissive temperature for pKD46 replication).
2. With a single colony inoculate the following:
1. Growth block well containing 1ml Freezing media + Kan(50ug/ml)/well .
2. LB+Kan agar in a 96 well plate
3. LB+Amp agar in a second 96-well plate
3. Grow overnight at 37°C. Cured cells will grow on Kan but not on Amp.
4. Confirm by PCR and sequencing as above.