Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
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__NOTOC__
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<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August2|August 2]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August11|August 11]]</div></div>
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==[[シングルコロニーアイソレーション]]==
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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>
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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
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* 実験中に沈殿が見られたため
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** コンピテントセルの溶解が不十分だった？
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** 混ぜ方が不十分だった？
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* 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？
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3-1E以外を新しい培地に移した
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==PCR反応液の調整==
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==Single Colony Isolation==
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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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* Observed if any colonies were made
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{|style="text-align: center; margin-left:20px"
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** Could not see [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
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* Number of other colonies on other plates was also very small
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** Mistake in protocol is inferred
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** Precipitation of cells maybe at fault
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* All but [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]] was moved to new plates
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==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
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Reaction system was prepared for 3 parts, 245 uL in total
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{|style="text-align: center; margin-left:20px" class="protocol"
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|-
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!Reagent
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!Amount
|-
|-
| Autoclaved DW
| Autoclaved DW
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| 5 uL
| 5 uL
|-
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''245 uL'''
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|style="border-top:1px solid #996;"|'''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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* 49 uL of reaction solution was added to each of 4 tubes and after DNA template was added
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* それぞれのPCRチューブとTemplate，lengthは
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* Length of each template is listed int the table below
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{|border="1" style="margin-left: 20px;"
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{|border="1" style="margin-left: 20px;" class="protocol"
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|-
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!Tube
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!Biobrick
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!Length
|-
|-
| 1
| 1
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| 2-24G（sender）
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
| 847 bp
| 847 bp
|-
|-
| 2
| 2
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| 1-2M（RBS）
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
| 61 bp
| 61 bp
|-
|-
| 3
| 3
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| 1-23L（terminator）
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
| 178 bp
| 178 bp
|-
|-
| 4
| 4
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| 3-1E（heat sensor）
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
| 984 bp
| 984 bp
|}
|}
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* DNA lengthはパーツ長＋プライマー(49 bp)
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* Template DNA length is　calculated by adding prefix and suffix length
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* 3-1EはTransformationがうまくいかなかったため
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** Biobrick Length + Prefix length + Suffix Length
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* Because [[Team:HokkaidoU_Japan/Protocols|Mini prep]] of 3-1E failed, it amplified by PCR
==PCR program==
==PCR program==
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{| border="1"  style="margin-left: 20px;"
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{|style="margin-left: 20px;" class="protocol"
|-
|-
| Predenature
| Predenature
| 94℃ 2 min
| 94℃ 2 min
|-
|-
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| Denature
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|style="border-top:1px solid #996;"| Denature
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| 98℃ 10 sec
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|style="border-top:1px solid #996;"| 98℃ 10 sec
|-
|-
| Extension
| Extension
| 68℃ 30 sec
| 68℃ 30 sec
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|-
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|style="border-top:1px solid #996;"| Hold
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|style="border-top:1px solid #996;"| 4℃
|}
|}
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* 30 cycle
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* 30 cycles
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* 30 sec/kbpなので１cycle30秒で行った
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* KOD potency is 30 sec/kbp, so 30 sec for １ cycle
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==[[アガロースゲル電気泳動|電気泳動]]==
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==Electrophoresis==
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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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[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|Electrophoresis of amplified BioBricks]]
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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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* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
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[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|short discription]]
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* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f1]
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* Added 20 uL of EtBr to 1/2 TBE
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{|border="1" style="margin-left: 20px;" class="protocol"
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!Lane
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!DNA
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!Length of DNA
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|-
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| 1
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| pUC119/''Hin''f1
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|
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|-
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| 2
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
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| 847 bp
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|-
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| 3
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
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| 61 bp
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|-
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| 4
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
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| 178 bp
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|-
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| 5
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
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| 984 bp
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|}

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"