Team:HokkaidoU Japan/Notebook/September29

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[[Image:HokkaidoU Japan 20100826a.jpg‎|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]
+
= Electrophoresis of Yesterday Samples =
 +
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.
-
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed.
+
[[Image:HokkaidoU Japan 20100929a.jpg|200px|center|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]
 +
 
 +
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =
 +
*PCRed 5 colony.
 +
 
 +
 
 +
PCR mix
 +
{|style="text-align: center" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|Quick Taq
 +
|25 uL
 +
|-
 +
|EX-F
 +
|0.5 uL
 +
|-
 +
|PS-R
 +
|0.5 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''26 uL'''
 +
|}
 +
 
 +
*PCRed according to the table below.98C and 68C for 35 cycles.
 +
 
 +
<br>
 +
{|style="text-align: center" class="protocol"
 +
!temp
 +
!time
 +
|-
 +
|94C
 +
|2 min
 +
|-
 +
|94C
 +
|30 sec
 +
|-
 +
|68C
 +
|90 sec
 +
|-
 +
|4C
 +
|hold
 +
|-
 +
|}
 +
 
 +
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.
 +
 
 +
[[Image:HokkaidoU Japan 20100929b.jpg|200px|center|thumb|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
 +
 
 +
<div style="float:left;">
 +
{|style="text-align: center" class="protocol"
 +
!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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''50 uL'''
 +
|}
 +
</div>
 +
<div style="float:left;">
 +
{|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'''
 +
|}
 +
</div>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
*PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.
 +
 
 +
<br>
 +
<div style="float:left;">
 +
{|style="text-align: center" class="protocol"
 +
!temp
 +
!time
 +
|-
 +
|94C
 +
|2 min
 +
|-
 +
|98C
 +
|10 sec
 +
|-
 +
|68C
 +
|60 sec
 +
|-
 +
|4C
 +
|hold
 +
|-
 +
|}
 +
</div>
 +
<div style="float:left;">
 +
{|style="text-align: center" class="protocol"
 +
!temp
 +
!time
 +
|-
 +
|94C
 +
|2 min
 +
|-
 +
|94C
 +
|30 sec
 +
|-
 +
|68C
 +
|90 sec
 +
|-
 +
|4C
 +
|hold
 +
|-
 +
|}
 +
</div>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
*Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid.

Latest revision as of 17:28, 27 October 2010

Notebook

Electrophoresis of Yesterday Samples

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

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
Reagent Amount
Quick Taq 7 uL
EX-F 1.5 uL
PS-R 1.5 uL
colony solution 10 uL
Total 20 uL















  • PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.


temp time
94C 2 min
98C 10 sec
68C 60 sec
4C hold
temp time
94C 2 min
94C 30 sec
68C 90 sec
4C hold









  • Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid.
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