Team:HokkaidoU Japan/Notebook/September14

From 2010.igem.org

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=pSB1C3, araC Promoter, GFPの制限酵素処理～Transformation=
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=Digestion and ligation of pSB1C3, araC Promoter and GFP
 +
 
==Digestion==
==Digestion==
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[[Image:HokkaidoU Japan 20100914a.jpg‎|200px|right|thumb|]]
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pSB1C3は9月6日にPCR後のゲル抽したDNA(2256 ng/uL)を20倍に希釈して使用した．
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[[Image:HokkaidoU Japan 20100914a.jpg‎|200px|right|thumb|Electrophoresed digestion product]]
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 +
Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.
 +
 
{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
!Reagent
!Reagent
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<div style="clear:both"></div>
<div style="clear:both"></div>
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→37℃で'''2時間'''培養<br>
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→Incubated at 37C for 120 min<br>
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→それぞれにSample Buffer 10 uLを加えて電気泳動
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→Added Sample Buffer 10 uL and electrophoresed
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* pSB1C3はバンドが確認できなかった
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* pSB1C3band wasn't visible
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→ほかの2つはゲル抽して回収
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→ Other 2 were gel extracted
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* 40 uLになった
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* Diluted that final volume would be 40 uL
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==エタ沈==
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==Ethanol precipitation==
[[Image:HokkaidoU Japan 20100914b.jpg‎|200px|right|thumb|]]
[[Image:HokkaidoU Japan 20100914b.jpg‎|200px|right|thumb|]]
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* 上のDNA solution 40 uLに4 uLの3M 酢酸ナトリウムを加えた
 
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* 44 uLの100% EtOHを加え，ドライアイスで凍結させた
 
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* 解凍15,996rpm@4℃で5分間遠心した
 
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* 上清を捨て，100 uLの70% EtOHでリンスし，同じく遠心
 
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* 上清を捨て，乾燥させて，それぞれ2 uLのTEで溶かした
 
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promoter: 125 ng/2uL，GFP: 106 ng/2uLになっているはず
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* Added ethanol acetate[3M] 4 uL to 40 uL of previously purified DNA solutuion
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* pSB1C3は9月6日にEPcut，エタ沈したものを電気泳動して濃度を確かめた
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* Added EtOH[100%] 44 uL friezed in dry ice
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* 20 ng/uLくらい
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* Melted and centrifuged at 15,996rpm, 4C 5 min
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→2 uL使う
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* Discarded supernatant and added EtOH[70%] 100 uL rinsed
 +
* Centrifuged at 15,996rpm, 4C 5 min
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* Discarded supernatant and dried
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* Diluted in 2 uL of TE each
 +
 
 +
Anticipated concentrations
 +
promoter: 125 ng/2uL
 +
GFP: 106 ng/2uL
 +
 
 +
* pSB1C3 was cut(EcoR1, Pst1) on September 6th
 +
* Did ethanol precipitation and electrophoresed to check concentration
 +
* It was about 20 ng/uL
 +
→Used 2 uL of it
==Ligation==
==Ligation==
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{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
!Reagent
!Reagent
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|}
|}
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このうち5 uLは先生がエレクトロポレーションに使用．残りを50 uLコンピに[[Transformation]]
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*50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)

Revision as of 11:15, 28 September 2010

Notebook

=Digestion and ligation of pSB1C3, araC Promoter and GFP

Digestion

Electrophoresed digestion product

Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.

Reagent Amount
10x M buffer 5 uL
DW 34
pSB1C3 1
BSA 5
EcoR I 2
Pst I 3
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 27
Promoter 8
BSA 5
EcoR I 3
Spe I 2
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 32
GFP 3
BSA 5
Xba I 4
Pst I 1
Total 50 uL

→Incubated at 37C for 120 min
→Added Sample Buffer 10 uL and electrophoresed

  • pSB1C3band wasn't visible

→ Other 2 were gel extracted

  • Diluted that final volume would be 40 uL

Ethanol precipitation

HokkaidoU Japan 20100914b.jpg
  • Added ethanol acetate[3M] 4 uL to 40 uL of previously purified DNA solutuion
  • Added EtOH[100%] 44 uL friezed in dry ice
  • Melted and centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and added EtOH[70%] 100 uL rinsed
  • Centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and dried
  • Diluted in 2 uL of TE each

Anticipated concentrations promoter: 125 ng/2uL GFP: 106 ng/2uL

  • pSB1C3 was cut(EcoR1, Pst1) on September 6th
  • Did ethanol precipitation and electrophoresed to check concentration
  • It was about 20 ng/uL

→Used 2 uL of it


Ligation

Reagent Amount
pSB1C3 2 uL
promoter 2 uL
GFP 2 uL
Mighty Mix 6 uL
T4 ligase 0.4 uL
Total 12.4 uL'
  • 50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)
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