Step 1 - Amplify ORF from MG1655
1.0 ul genomic DNA (30ng/ul)
95°C 95°C 55°C
72°C 72°C 4°C
Step 2 - Transposition reaction
6 ul ORF PCR product (~100-200ng)
Vortex, spin down
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
2. Transfer entire volume (~45ul) to chilled cubette (0.1cm gap), kept on ice
3. Electroporate with the following settings (for BioRad Pulse Controller)
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
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.
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
2. PCR Reaction.
5 ul diluted culture
95°C 94°C 55°C 72°C
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
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:
3. Grow overnight at 37°C. Cured cells will grow on Kan but not on Amp.
4. Confirm by PCR and sequencing as above.
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