• glycerol-stock of E.coli with salmonella's BAC library vector
• making competent cell of E.coli with SPI2
• plasmid & GFP-double terminator's Ligation & Transformation
• PCR of E.coli with T3SSsignal and of GFP-double terminator

Ligation of plasmid and GFP-double terminator & Transformation

1. added 2 uL TE into plasmid solvant and GFP-double terminator solvant
2. mixed the samples
3. added 5 uL Mighty mix
4. incubated at 16C for 30min
5. added the sample to 100 uL competent cell
6. incubated at 0C for 30min
7. heatshocked at 42C for 60sec
8. incubated at 0C for 5min
9. added sample to 400 uL LB
10. incubated at 37C for 2 hours
11. plated the sample on LBA medium
12. incubated at 37C

PCR of Parts

• We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.

• PCRed according to the table below.98C and 68C were 45 cycles.

• electrophoresed the two sample, but a band of T3SS signal wasn't seen.PCRed and electrophoresed again,but a band of T3SS signal wasn't seen clearly.それらしい影を切り出してゲル抽出した。