Team:HokkaidoU Japan/Notebook/September29

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Notebook

Electrophoresis of Yesterday Samples

Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)
  • electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.

















Colony PCR of Arabinose Promoter + RBS + GFP + double terminator

  • PCRed 5 colony.


PCR mix

Reagent Amount
Quick Taq 25 uL
EX-F 0.5 uL
PS-R 0.5 uL
Total 26 uL
  • PCRed according to the table below.98C and 68C for 35 cycles.


temp time
94C 2 min
94C 30 sec
68C 90 sec
4C hold
  • erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.
Electrophoresis of colony PCR sample















Cultivation of colony

  • cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.

PCR of T3SS signal again

  • We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.

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
{|style="text-align: center" class="protocol"

!Reagent !Amount |- |Quick Taq |7 uL |- |EX-F |1.5 uL |- |PS-R |1.5 uL |- |colony solution |10 uL |- |style="border-top:1px solid #996"|Total |style="border-top:1px solid #996"|20 uL |}

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