Team:HokkaidoU Japan/Notebook/August24

From 2010.igem.org

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(→ラオリプライマーで増やしたパーツの確認)
 
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* This vector didn't have a primer annealing site for our new primers
* This vector didn't have a primer annealing site for our new primers
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=Ligation Mixtureのチェック=
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=Check of Ligation Mixtures=
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Ligation Mixture「I」とLigation Mixture「M」について
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TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M]
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* PCRで増やしてゲル抽したpSB1C3 2 uLにLigation Mixture 4 uLをいれ，それぞれのTransformationした
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* After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added
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* LB 200 uLを加え，100 uLずつをChloramphenicol 34 ug/uLと 12 ug/uLの培地にまいた
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* Transformed
 +
* incubated in 200 uL of LB medium
 +
* 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol
 +
 
 +
=Various Checks=
 +
 
 +
[[Image:HokkaidoU Japan 20100824b.jpg‎|200px|right|thumb|Electrophoresis to check if various DNA manipulations were successful]]
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=いろいろ確認=
 
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[[Image:HokkaidoU Japan 20100824b.jpg‎|200px|right|thumb|]]
 
{| class="protocol"
{| class="protocol"
|'''Lane'''
|'''Lane'''
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|-
|-
|2
|2
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|λ/''Hin''d III
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]
|-
|-
|3
|3
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|pSB1C3どうしのLigation
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|Ligation between pSB1C3 vectors
|-
|-
|4
|4
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|ラオリプライマーによる1-2NのPCRのろ過産物（上）
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|Product obtained by using digestion visualization primer for 1-2N
|-
|-
|5
|5
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|ラオリプライマーによる1-2NのPCRのろ過（下）
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|Flow through of digestion visualization primer for 1-2N
|-
|-
|6
|6
-
|λ/''Hin''d III
+
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]
|-
|-
|7
|7
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|ゲル抽したpSB1C3
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|Gel extracted pSB1C3
|}
|}
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レーン３でバンドが見られなかった．
+
Band weren't visible in lane 3
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* ダイマー，テトラマーなど，さまざまな断片ができ，薄くなってしまった？
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* Dimers, trimers and etc were hardly visible maybe severely diluted
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レーン７は薄いがバンドが確認できた
+
 
-
* ゲル抽に問題はない
+
In lane 7 had detectable by very weak band
 +
* There weren't any problems with gel extraction

Latest revision as of 07:44, 27 October 2010

Notebook

Check to see if digestion visualization Primers Work

Electrophoresis after restriction enzyme digestion‎
  • All 4 of PCR products were purified via Microcon YM-10
Lane DNA
1 TSUDA Marker I
3 RBS
4 GFP
5 double terminator
6 Promoter

Promoter band in lane 6 wasn't visible

  • This vector didn't have a primer annealing site for our new primers

Check of Ligation Mixtures

TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M]

  • After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added
  • Transformed
  • incubated in 200 uL of LB medium
  • 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol

Various Checks

Electrophoresis to check if various DNA manipulations were successful
Lane DNA
2 λ/Hind III
3 Ligation between pSB1C3 vectors
4 Product obtained by using digestion visualization primer for 1-2N
5 Flow through of digestion visualization primer for 1-2N
6 λ/Hind III
7 Gel extracted pSB1C3

Band weren't visible in lane 3

  • Dimers, trimers and etc were hardly visible maybe severely diluted

In lane 7 had detectable by very weak band

  • There weren't any problems with gel extraction
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