Team:HokkaidoU Japan/Notebook/September28


  • glycerol-stock of E.coli with salmonella's BAC library vector
  • making of competent cell of E.coli with SPI2 BAC vector
  • 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 which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:


Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:


PCR mix

Reagent Amount
colony solution 10 uL
DW 23 uL
10x PCR Buffer 5 uL
5 mM 4dNTPs 5 uL
25 mM MgSO4 3 uL
EX-RBS Primer 1.5 uL
SlrP3 Primer 1.5 uL
KOD plus neo 1 uL
Total 50 uL
Reagent Amount
1-12K 1 uL
DW 32.5 uL
10x PCR Buffer 5 uL
5 mM 4dNTPs 5 uL
25 mM MgSO4 3 uL
NLS Primer 1.5 uL
PS-R 1.5 uL
KOD plus neo 1 uL
Total 50 uL

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

temp time
94C 2 min
98C 10 sec
68C 60 sec
4C hold
  • electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.
Stored at -20C.