Team:HokkaidoU Japan/Notebook/August10

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(→シングルコロニーアイソレーション)
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* Because Mini prep of 3-1E failed, it amplified by PCR
* Because 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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==
 
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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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|-
 
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| Autoclaved DW
 
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| 165 uL
 
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|-
 
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| 10x PCR buffer
 
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| 25 uL
 
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|-
 
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| 2 mM dNTPs
 
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| 25 uL
 
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|-
 
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| 25 mM MgSO<sub>4</sub>
 
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| 15 uL
 
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|-
 
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| EX-F primer
 
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| 5 uL
 
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|-
 
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| PS-R primer
 
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| 5 uL
 
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|-
 
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| KOD plus Neo
 
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| 5 uL
 
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|-
 
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|style="border-top:1px solid #000;"|'''Total'''
 
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|style="border-top:1px solid #000;"|'''245 uL'''
 
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|}
 
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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
 
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{|border="1" style="margin-left: 20px;" class="protocol"
 
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|-
 
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| 1
 
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| 2-24G（sender）
 
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| 847 bp
 
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|-
 
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| 2
 
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| 1-2M（RBS）
 
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| 61 bp
 
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|-
 
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| 3
 
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| 1-23L（terminator）
 
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| 178 bp
 
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|-
 
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| 4
 
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| 3-1E（heat sensor）
 
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| 984 bp
 
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|}
 
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* 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==

Revision as of 05:51, 20 September 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 for１cycle

Electrophoresis

Electrophoresis of amplified BioBricks
  • Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
  • Used marker pUC1d19/Hinf1
  • Added 20 uL of EtBr to 1/2 TBE
Lane DNA Length of DNA
1 pUC1d19/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

電気泳動

  • 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
  • マーカーはpUC119/Hinf1を使用
  • エチブロは20 uL使用
short discription
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