Team:HokkaidoU Japan/Notebook/September17

From 2010.igem.org

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Revision as of 07:09, 20 September 2010

Notebook
  • Construction of GFP marker for a part which will be secreted using T3SS
  • Ordered primers for construction for same part

Digestion of GFP and Double Terminator

Parts Information

Description BioBrick No. Well No. Length Plasmid
GFP BBa_E0040 1-14K 720bp pSB1A3
double terminator BBa_B0015 1-23L 129bp pSB1AK3
pSB1A3 pSB1A3 2157bp pSB1A3


Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.


  • Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
  • Estimated concentration from photo of electrophoresis
  • pSB1A3 solution was done by other person.
  • Made digestion recipes(below) based on estimated concentrations
  • Made 30ul of pSB1A3 solution, but latter found it insufficient to ligate parts
    • made more 50ul of it after


Part Well No. Amount
1-14K 200 ng/ul
1-23L 120 ng/ul
pSB1A3 2.5 ng/ul

Digestion

Reagent Amount
1-23L 0.5 uL
10x M buffer 5 uL
0.1%BSA 5 uL
Xba I 4 uL
Pst I 0.5 uL
DW 35 uL
Total 50 uL
Reagent Amount
1-14K 1.5 uL
10x H buffer 2 uL
0.1% BSA 2 uL
EcoR I 1 uL
Spe I 0.5 uL
DW 13 uL
Total 20 uL
Reagent Amount
pSB1A3 20 uL
10x H buffer 3 uL
0.1% BSA 3 uL
EcoR I 0.5 uL
Pst I 0.5 uL
DW 3 uL
Total 30 uL
Reagent Amount
pSB1A3 30 uL
10x H buffer 5 uL
0.1% BSA 5 uL
EcoR I 0.5 uL
Pst I 0.5 uL
DW 9 uL
Total 30 uL


  • Incubated each solution at 37C
  • Solution of 1-23L was incubated for 150 min
  • Solution of 1-14K was incubated for 90 min
  • 30ul of pSB1A3 solution was incubated for 60 min
  • 50ul of pSB1A3 solution was incubated for 30 min
  • Performed electrophoresis for each solution
  • put 12uls each into wells of a gel like below.
Lane DNA
1 λ/HindIII,　EcoR I
2~3 1-14K
4~8 1-23L
9~16 pSB1A3

Results

  • Could not see bands of 1-23L because leaked out
    • Drove current for too long
  • Extracted the other samples from a gel using promega kit
  • Stored all at -20C.
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