Team:HokkaidoU Japan/Notebook/September29
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= Electrophoresis of Yesterday Samples = | = Electrophoresis of Yesterday Samples = |
Revision as of 16:56, 27 October 2010
Electrophoresis of Yesterday Samples
- 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.
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 |}