Team:HokkaidoU Japan/Notebook/August25

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}
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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August24|August 24]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August26|August 26]]</div></div>
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=Transformationの結果=
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=Transformation Results=
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* M 34：コロニー無し
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==Colony Check==
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* M 12：コロニー有り
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* I 34：コロニー無し
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{| class="protocol"
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* I 12：コロニー有り
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|'''Plate'''
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* 1-3A 34：コロニー有り
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|'''Colonies'''
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* 1-3A 12：コロニー有り
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|-
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|M 34
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|no
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|-
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|M 12
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|yes
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|-
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|I 34
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|no
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|-
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|I 34
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|yes
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|-
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] 34
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|yes
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|-
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] 12
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|yes
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|}
<hr width="50%">
<hr width="50%">
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* 1-3A 34でコロニーが観察されたため，培地に問題はなさそう
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* [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
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* 12の培地は後からChloramphenicolを足したため，濃度にムラができたか
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** So the medium and antibiotic consentration seems ok,
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* 1-3A 12とは明らかに違うコロニーだった．
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* Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
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→Ligationの問題のほか，Transformationに使うDNAの濃度に問題があるかもしれない．
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* Colonies of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] and 12 were obviously different
 +
→ So we suspected issues in Ligation and DNA concentration bused for transformation
 +
 
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=Estimation of 1-3A Consentration=
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[[Image:HokkaidoU Japan 20100825a.jpg‎|200px|right|thumb|Electrophoresis of 1uL of 1-3A DNA]]
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=1-3AのDNA量推定=
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*Added 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] into 30 uL of competent cells
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[[Image:HokkaidoU Japan 20100825a.jpg‎|200px|right|thumb|]]
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*Transformation was successful
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1-3A 1 uLをコンピテントセル30 uLに溶かし，Transformationがうまくいった．
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*Electrophoresed 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]]，calculated amount of transformed DNA
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* 1-3A 1 uLを電気泳動し，TransformationできたDNA量を求める．
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→Estimated concentration 2 ng/uL
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→2 ng/uLと推定
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<div style="clear:both"></div>
<div style="clear:both"></div>
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=Ethanol precipitation of pSB1C3 and Ligation=
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=pSB1C3をエタ沈で濃縮，Ligation=
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===Ethanol precipitation===
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===エタ沈===
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# 3 M 酢酸ナトリウム 8.2 uL加える
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# Added 8.2 uL of sodium acetate [3 M]
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# 100%エタノールを205 uL加え，混ぜる
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# Added 205 uL of Ethanol [100%] and mixed
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# 15,00rpm@4℃で10分間遠心する
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# Centrifuge at 15000rpm, 4C for 10 min
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# 上清を捨て，500 uL 70%エタノールを加え，混ぜる
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# Discarded supernatant and added 500 uL of Ethanol [70%], mixed
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# 15,00rpm@4℃で5分間遠心する
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# Centrifuge at 15000rpm, 4C for 5 min
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# 上清を捨て，真空デシケータで乾燥させる
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# Discarded supernatant
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# DW 8.2 uLを加え，元の10倍に濃縮する
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# Dried via vacuum desiccator
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# Added 8.2 uL of ADW
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* Concentration increased 10 fold
===Ligation===
===Ligation===
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# 8.2 uL DNA solutionに同量のLigation Solutionを加える
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# Mixed 8.2 uL of DNA solution with the same volume of Ligation Solution
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# T4 ligase 1 uLを加え，16℃で30分間インキュベート
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# Added extra 1 uL of T4 ligase
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# 明日，電気泳動・ゲル抽・Transformation
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# Incubated at 16C for 30 min
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Tomorrow will do aditional Electrophoresis gel extraction and transformation
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=Digestion of PCR products amplified using digestion visualization primers=
 +
 
 +
 
 +
[[Image:HokkaidoU Japan 20100825c.JPG‎ |200px|right|thumb|Electrophoresis after digestion, differences in length is visible]]
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Calculated the concentration of DNA amplified yesterday
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=らおりプライマーで作ったPCR productsのDigestion=
 
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[[Image:HokkaidoU Japan 20100825c.JPG‎ |200px|right|thumb|]]
 
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前日のPCRから，増やすことのできたパーツのDNA量を求めた
 
{|
{|
|-
|-
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|}
|}
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===Digestion 反応系===
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===Digestion Reaction===
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{|style="text-align:center;"
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{|style="text-align:center;" class="protocol"
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!Reagent
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!Amount
|-
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|10x M buffer
|10x M buffer
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|0.5
|0.5
|-
|-
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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;"|'''10 uL'''
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|style="border-top:1px solid #996;"|'''10 uL'''
|}
|}
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→37℃で60分
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→ラオリプライマーの真価を電気泳動で確認
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→Incubated at 37C for 60 min
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→Confirmed how awesome new primers are

Latest revision as of 07:50, 27 October 2010

Notebook

Transformation Results

Colony Check

Plate Colonies
M 34 no
M 12 yes
I 34 no
I 34 yes
1-3A 34 yes
1-3A 12 yes
  • 1-3A had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
    • So the medium and antibiotic consentration seems ok,
  • Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
  • Colonies of 1-3A and 12 were obviously different

→ So we suspected issues in Ligation and DNA concentration bused for transformation

Estimation of 1-3A Consentration

Electrophoresis of 1uL of 1-3A DNA
  • Added 1 uL of 1-3A into 30 uL of competent cells
  • Transformation was successful
  • Electrophoresed 1 uL of 1-3A，calculated amount of transformed DNA

→Estimated concentration 2 ng/uL

Ethanol precipitation of pSB1C3 and Ligation

Ethanol precipitation

  1. Added 8.2 uL of sodium acetate [3 M]
  2. Added 205 uL of Ethanol [100%] and mixed
  3. Centrifuge at 15000rpm, 4C for 10 min
  4. Discarded supernatant and added 500 uL of Ethanol [70%], mixed
  5. Centrifuge at 15000rpm, 4C for 5 min
  6. Discarded supernatant
  7. Dried via vacuum desiccator
  8. Added 8.2 uL of ADW
  • Concentration increased 10 fold

Ligation

  1. Mixed 8.2 uL of DNA solution with the same volume of Ligation Solution
  2. Added extra 1 uL of T4 ligase
  3. Incubated at 16C for 30 min

Tomorrow will do aditional Electrophoresis gel extraction and transformation

Digestion of PCR products amplified using digestion visualization primers

Electrophoresis after digestion, differences in length is visible

Calculated the concentration of DNA amplified yesterday


RBS 27 ng/uL 312 bp
GFP 54 ng/uL 1020 bp
dT 49 ng/uL 429 bp

Digestion Reaction

Reagent Amount
10x M buffer 1 uL
DW 4.5
DNA 2.5
BSA 1
EcoR I 0.5
Pst I 0.5
Total 10 uL

→Incubated at 37C for 60 min

→Confirmed how awesome new primers are
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