Team:HokkaidoU Japan/Notebook/September29

From 2010.igem.org

(Difference between revisions)
Line 83: Line 83:
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.
-
[[Image:HokkaidoU Japan 20100929b.jpg|200px|left|thumb|Electrophoresis of colony PCR sample]]
+
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]
Line 116: Line 116:
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.
*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>

Revision as of 16:54, 27 October 2010

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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