Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}
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==Single Colony Isolation==
==Single Colony Isolation==
* Observed if any colonies were made
* Observed if any colonies were made
-
** Could not see 3-1E
+
** Could not see [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
* Number of other colonies on other plates was also very small
* Number of other colonies on other plates was also very small
** Mistake in protocol is inferred
** Mistake in protocol is inferred
** Precipitation of cells maybe at fault
** Precipitation of cells maybe at fault
-
* All but 3-1E was moved to new plates
+
* All but [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]] was moved to new plates
-
==シングルコロニーアイソレーション==
+
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
-
コロニーを確認したところ，3-1Eにはコロニーが見られなかった<br>
+
-
ほかのプレートもコロニー数が少なかったため，手順に不完全なところがあったのかもしれない
+
-
* 実験中に沈殿が見られたため
+
-
** コンピテントセルの溶解が不十分だった？
+
-
** 混ぜ方が不十分だった？
+
-
* ２回目の実験で，若干急いだ感じだったので，吸着などが不十分だった？
+
-
3-1E以外を新しい培地に移した
+
-
 
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-
==PCR Reaction System==
+
Reaction system was prepared for 3 parts, 245 uL in total
Reaction system was prepared for 3 parts, 245 uL in total
Line 49: Line 40:
| 5 uL
| 5 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
+
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''245 uL'''
+
|style="border-top:1px solid #996;"|'''245 uL'''
|}
|}
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|-
|-
| 1
| 1
-
| 2-24G（sender）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
| 847 bp
| 847 bp
|-
|-
| 2
| 2
-
| 1-2M（RBS）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
| 61 bp
| 61 bp
|-
|-
| 3
| 3
-
| 1-23L（terminator）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
| 178 bp
| 178 bp
|-
|-
| 4
| 4
-
| 3-1E（heat sensor）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
| 984 bp
| 984 bp
|}
|}
* Template DNA length is　calculated by adding prefix and suffix length
* Template DNA length is　calculated by adding prefix and suffix length
** Biobrick Length + Prefix length + Suffix Length
** Biobrick Length + Prefix length + Suffix Length
-
* Because Mini prep of 3-1E failed, it amplified by PCR
+
* Because [[Team:HokkaidoU_Japan/Protocols|Mini prep]] of 3-1E failed, it amplified by PCR
-
 
+
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==PCR反応液の調整 ==
+
-
==
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今回は4パーツ増幅のためTotal 245 uL作成した．
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-
{|style="text-align: center; margin-left:20px" class="protocol"
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-
|-
+
-
| Autoclaved DW
+
-
| 165 uL
+
-
|-
+
-
| 10x PCR buffer
+
-
| 25 uL
+
-
|-
+
-
| 2 mM dNTPs
+
-
| 25 uL
+
-
|-
+
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| 25 mM MgSO<sub>4</sub>
+
-
| 15 uL
+
-
|-
+
-
| EX-F primer
+
-
| 5 uL
+
-
|-
+
-
| PS-R primer
+
-
| 5 uL
+
-
|-
+
-
| KOD plus Neo
+
-
| 5 uL
+
-
|-
+
-
|style="border-top:1px solid #000;"|'''Total'''
+
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|style="border-top:1px solid #000;"|'''245 uL'''
+
-
|}
+
-
 
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* 反応液49 uLを４本のPCRチューブに分注し，Templateを1 uLずつ加えた
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* それぞれのPCRチューブとTemplate，lengthは
+
-
{|border="1" style="margin-left: 20px;" class="protocol"
+
-
|-
+
-
| 1
+
-
| 2-24G（sender）
+
-
| 847 bp
+
-
|-
+
-
| 2
+
-
| 1-2M（RBS）
+
-
| 61 bp
+
-
|-
+
-
| 3
+
-
| 1-23L（terminator）
+
-
| 178 bp
+
-
|-
+
-
| 4
+
-
| 3-1E（heat sensor）
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-
| 984 bp
+
-
|}
+
-
* DNA lengthはパーツ長＋プライマー(49 bp)
+
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* 3-1EはTransformationがうまくいかなかったため
+
==PCR program==
==PCR program==
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|}
|}
* 30 cycles
* 30 cycles
-
* KOD potency is 30 sec/kbp, so 30 sec for１cycle
+
* KOD potency is 30 sec/kbp, so 30 sec for １ cycle
==Electrophoresis==
==Electrophoresis==
-
[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|Electrophoresis of amplified BioBricka]]
+
[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|Electrophoresis of amplified BioBricks]]
* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
-
* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC1d19/''Hin''f1]
+
* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f1]
* Added 20 uL of EtBr to 1/2 TBE
* Added 20 uL of EtBr to 1/2 TBE
Line 166: Line 104:
|-
|-
| 1
| 1
-
| pUC1d19/''Hin''f1
+
| pUC119/''Hin''f1
|  
|  
|-
|-
| 2
| 2
-
| 2-24G（sender）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
| 847 bp
| 847 bp
|-
|-
| 3
| 3
-
| 1-2M（RBS）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
| 61 bp
| 61 bp
|-
|-
| 4
| 4
-
| 1-23L（terminator）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
| 178 bp
| 178 bp
|-
|-
| 5
| 5
-
| 3-1E（heat sensor）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
| 984 bp
| 984 bp
|}
|}
-
 
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==電気泳動==
 
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* 増幅したDNAを5 uLとって6xサンプルバッファー1 uLを加えた
 
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* マーカーはpUC119/''Hin''f1を使用
 
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* エチブロは20 uL使用
 
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[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|short discription]]
 

Latest revision as of 07:14, 27 October 2010

Notebook

Single Colony Isolation

  • Observed if any colonies were made
    • Could not see 3-1E
  • Number of other colonies on other plates was also very small
    • Mistake in protocol is inferred
    • Precipitation of cells maybe at fault
  • All but 3-1E was moved to new plates

PCR Reaction System

Reaction system was prepared for 3 parts, 245 uL in total

Reagent Amount
Autoclaved DW 165 uL
10x PCR buffer 25 uL
2 mM dNTPs 25 uL
25 mM MgSO4 15 uL
EX-F primer 5 uL
PS-R primer 5 uL
KOD plus Neo 5 uL
Total 245 uL
  • 49 uL of reaction solution was added to each of 4 tubes and after DNA template was added
  • Length of each template is listed int the table below
Tube Biobrick Length
1 2-24G（sender） 847 bp
2 1-2M（RBS） 61 bp
3 1-23L（terminator） 178 bp
4 3-1E（heat sensor） 984 bp
  • Template DNA length is　calculated by adding prefix and suffix length
    • Biobrick Length + Prefix length + Suffix Length
  • Because Mini prep of 3-1E failed, it amplified by PCR

PCR program

Predenature 94℃ 2 min
Denature 98℃ 10 sec
Extension 68℃ 30 sec
Hold 4℃
  • 30 cycles
  • KOD potency is 30 sec/kbp, so 30 sec for １ cycle

Electrophoresis

Electrophoresis of amplified BioBricks
  • Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
  • Used marker pUC119/Hinf1
  • Added 20 uL of EtBr to 1/2 TBE
Lane DNA Length of DNA
1 pUC119/Hinf1
2 2-24G（sender） 847 bp
3 1-2M（RBS） 61 bp
4 1-23L（terminator） 178 bp
5 3-1E（heat sensor） 984 bp
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August10"