Team:HokkaidoU Japan/Notebook/October2

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{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
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<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/October1|October 1]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/October3|October 3]]</div></div>
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[[Image:HokkaidoU Japan 20101002a.JPG|200px|right|thumb|Fig.1]]
==Colony PCR==
==Colony PCR==
-
[[Image:HokkaidoU Japan 20101002a.JPG|200px|right|thumb]]
+
* Performed Colonie PCR for 3 colonies which were incubated over night (Fig.1)
-
* Performed Colonie PCR for 3 colonies which were incubated over night
+
* Colony numbers were: 1, 2 and 3
* Colony numbers were: 1, 2 and 3
* Results showed no insertion  
* Results showed no insertion  
** but when result came, had already done miniprep and prepared Sequencing Master Mix
** but when result came, had already done miniprep and prepared Sequencing Master Mix
<div style="clear:both;"></div>
<div style="clear:both;"></div>
 +
==[[Team:HokkaidoU_Japan/Protocols|Miniprep]]==
==[[Team:HokkaidoU_Japan/Protocols|Miniprep]]==
* Used Qiagen kit
* Used Qiagen kit
Line 36: Line 37:
|16.5
|16.5
|-
|-
-
|H2O
+
|DW
|5 uL
|5 uL
|80 uL
|80 uL
Line 45: Line 46:
|}
|}
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
=3 Piece Ligationやり直し=
+
----
-
[[Image:HokkaidoU Japan 20101002b.JPG|200px|right|thumb]]
+
 
-
==ゲル抽==
+
==3 Piece Ligation: Retry==
-
10月1日に制限酵素処理したDNA solutionをゲル抽
+
===[[Team:HokkaidoU_Japan/Protocols|Gel Extraction]]===
-
*左からλ/''Hin''dIII, pSB1T3, pSB1A3, Arabinose Promoter([[Team:HokkaidoU_Japan/Material_And_Methods#Materials_And_Methods|3-20B]]), T3SS signal, T3SS signal
+
Gel Extraction of DNAs that had been cut on October 1
-
** T3SS signalはバンドが見えないため，制限酵素処理からやり直し．原因不明．
+
* Couldn't see T3SS signal's bands, retried digestion like below 
 +
===Digestion: Retry===
 +
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|10 uL
 +
|-
 +
|DW
 +
|64
 +
|-
 +
|T3SS signal
 +
|6
 +
|-
 +
|BSA
 +
|10
 +
|-
 +
|Xba I
 +
|9.6
 +
|-
 +
|Pst I
 +
|0.4
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''100 uL'''
 +
|}
 +
-> Electrophoresed with other samples
 +
 
 +
----
 +
 
 +
==2 piece ligation of T3SS signal and pSB1C3 for DNA Submission==
 +
[[Image:HokkaidoU Japan 20101002b.JPG|200px|right|thumb|Fig.2]]
 +
<div style="float:left;">
 +
'''T3SS signal'''
 +
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|2 uL
 +
|-
 +
|DW
 +
|12
 +
|-
 +
|DNA
 +
|3
 +
|-
 +
|BSA
 +
|2
 +
|-
 +
|EcoR I
 +
|0.5
 +
|-
 +
|Pst I
 +
|0.5
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''20 uL'''
 +
|}
 +
</div>
 +
<div style="float:left; margin-left:50px;">
 +
'''pSB1C3'''
 +
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|2 uL
 +
|-
 +
|DW
 +
|13
 +
|-
 +
|DNA
 +
|2
 +
|-
 +
|BSA
 +
|2
 +
|-
 +
|EcoR I
 +
|0.5
 +
|-
 +
|Pst I
 +
|0.5
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''20 uL'''
 +
|}
 +
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
 +
->Incubated at 37C for 2 hrs
 +
* Electrophoresis (Fig.2: λ/''Hin''dIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells))
-
=Colony PCR=
+
<div style="clear:both;"></div>
-
[[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb]]
+
 
-
[[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb]]
+
----
-
[[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb]]
+
[[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb|Fig.3]]
 +
==Colony PCR (previous day's 3 piece ligation)==
 +
Did colony PCR of latecomers (No.5 to No.16)(Fig.3)
 +
* No.8, 10, 11, 12, 15 looked like good
 +
* Transfered No.8, 10, 11, 12, 13, 14, 15, 16(control) to 2 mL LBT (Tetracycline 15 ug/mL) and started incubation
 +
<div style="clear:both;"></div>
 +
 
 +
----
 +
 
 +
[[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb|Fig.4]]
 +
[[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb|Fig.5]]
 +
==Colony PCR (T3SS signal)==
 +
Did colony PCR to amplify T3SS signal (from BAC clone that has T3SS signal insertion)
 +
* Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
 +
* Extention: 90 sec, 40 cycles
 +
Electrophoresed to purify the DNA via gel extraction (Fig.4)<br>
 +
Electrophoresed to estimate the concentration of the DNA (Fig.5: λ/''Hin''dIII 2 uL, DNA solution 1 uL)
 +
* Concentration was estimated at 40 ng/uL
 +
<div style="clear:both;"></div>
 +
 
 +
===Digestion===
 +
[[Image:HokkaidoU Japan 20101002f.JPG|200px|right|thumb|Fig.6]]
 +
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x H buffer
 +
|2 uL
 +
|-
 +
|DW
 +
|9
 +
|-
 +
|DNA
 +
|5
 +
|-
 +
|BSA
 +
|2
 +
|-
 +
|EcoR I
 +
|1
 +
|-
 +
|Pst I
 +
|1
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''20 uL'''
 +
|}
 +
->Electrophoresis, Gel Extraction (Fig.6)

Latest revision as of 12:51, 26 October 2010

Notebook
Fig.1

Colony PCR

  • Performed Colonie PCR for 3 colonies which were incubated over night (Fig.1)
  • Colony numbers were: 1, 2 and 3
  • Results showed no insertion
    • but when result came, had already done miniprep and prepared Sequencing Master Mix

Miniprep

  • Used Qiagen kit
  • Melted in 50 uL TE instead of H2O

Preparation for Sequencing

  • Mixed as shown in the table below


5x Sequencing Buffer 1.5uL 24.75 uL Ready Reaction Premix 1 uL 16.5 H2O 5 uL 80 uL total 7.5/sample

Reagent Amount Amount for 16.5
5x Sequencing Buffer 1.5uL 24.75 uL
Ready Reaction Premix 1 uL 16.5
DW 5 uL 80 uL
Total 7.5/sample 121.25

3 Piece Ligation: Retry

Gel Extraction

Gel Extraction of DNAs that had been cut on October 1

  • Couldn't see T3SS signal's bands, retried digestion like below

Digestion: Retry

Reagent Amount
10x M buffer 10 uL
DW 64
T3SS signal 6
BSA 10
Xba I 9.6
Pst I 0.4
Total 100 uL

-> Electrophoresed with other samples

2 piece ligation of T3SS signal and pSB1C3 for DNA Submission

Fig.2

T3SS signal

Reagent Amount
10x M buffer 2 uL
DW 12
DNA 3
BSA 2
EcoR I 0.5
Pst I 0.5
Total 20 uL

pSB1C3

Reagent Amount
10x M buffer 2 uL
DW 13
DNA 2
BSA 2
EcoR I 0.5
Pst I 0.5
Total 20 uL

->Incubated at 37C for 2 hrs

  • Electrophoresis (Fig.2: λ/HindIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells))
Fig.3

Colony PCR (previous day's 3 piece ligation)

Did colony PCR of latecomers (No.5 to No.16)(Fig.3)

  • No.8, 10, 11, 12, 15 looked like good
  • Transfered No.8, 10, 11, 12, 13, 14, 15, 16(control) to 2 mL LBT (Tetracycline 15 ug/mL) and started incubation
Fig.4
Fig.5

Colony PCR (T3SS signal)

Did colony PCR to amplify T3SS signal (from BAC clone that has T3SS signal insertion)

  • Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
  • Extention: 90 sec, 40 cycles

Electrophoresed to purify the DNA via gel extraction (Fig.4)
Electrophoresed to estimate the concentration of the DNA (Fig.5: λ/HindIII 2 uL, DNA solution 1 uL)

  • Concentration was estimated at 40 ng/uL

Digestion

Fig.6
Reagent Amount
10x H buffer 2 uL
DW 9
DNA 5
BSA 2
EcoR I 1
Pst I 1
Total 20 uL
->Electrophoresis, Gel Extraction (Fig.6)
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/October2"