Team:HokkaidoU Japan/Notebook/August12

From 2010.igem.org

(Difference between revisions)
(Electrophoresis 1)
 
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* Checked the result of yesterdays ligation of PCR products by electrophoresis
* Checked the result of yesterdays ligation of PCR products by electrophoresis
* Added 4 uL of 6x Sample Buffer to make 12 uL in total
* Added 4 uL of 6x Sample Buffer to make 12 uL in total
-
* Used 20uL of EtOh
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* Used 20 uL of EtOH
<div style="clear:both"></div>
<div style="clear:both"></div>
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=mini prep=
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=[[Team:HokkaidoU_Japan/Protocols|Mini prep]]=
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* LB液体培地で18時間培養した菌を使用した
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* Used cells incubated for 18h in LB broth
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** 1-18Fは生育が悪かった
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** [[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]] didn't grow well
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* Chloroformのあと遠心し、430 uLの上清をとった
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* Centrifuge after adding 430 uL of chloroform、collected supernatant
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* 1-18Fは2-プロパノールを入れたあと,遠心しても沈殿がほとんど得られなかった
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* Added 2-propanol to [[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]], but after centrifugation there were no precipitation
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* 最後に30 uLのTEに溶かし、電気泳動をした
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** Melted in 30 uL of TE and did electrophoresis
 +
 
 +
=Electrophoresis 2=
 +
 
 +
[[Image:HokkaidoU Japan 20100812b.jpg‎|200px|right|thumb|Take 1]]
 +
[[Image:HokkaidoU Japan 20100812c.jpg‎|200px|right|thumb|Take 2 after few minutes]]
 +
 
 +
Checked plasmids gathered via [[Team:HokkaidoU_Japan/Protocols|mini prep]] and [[Team:HokkaidoU_Japan/Protocols|PCR]] products, by electrophoresis. <br>
 +
 
 +
 
 +
Because we wont do restriction enzyme digestion reaction system is ass follows.
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=電気泳動2=
 
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[[Image:HokkaidoU Japan 20100812b.jpg‎|200px|right|thumb|撮影1回目]]
 
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[[Image:HokkaidoU Japan 20100812c.jpg‎|200px|right|thumb|撮影2回目]]
 
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mini prepで得たプラスミドとPCRで得たパーツのDNAを電気泳動で確認した<br>
 
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制限酵素処理はしないので、反応系は以下の通り
 
{| style="text-align:center" class="protocol"
{| style="text-align:center" class="protocol"
|-
|-
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|style="border-bottom:1px solid #000; border-right:1px solid #000;"|
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|style="border-bottom:1px solid #996; border-right:1px solid #996;"|'''Lane'''
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|style="border-bottom:1px solid #000;"| 1
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|style="border-bottom:1px solid #996;"| 1
-
|style="border-bottom:1px solid #000;"| 2
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|style="border-bottom:1px solid #996;"| 2
-
|style="border-bottom:1px solid #000;"| 3
+
|style="border-bottom:1px solid #996;"| 3
-
|style="border-bottom:1px solid #000;"| 4
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|style="border-bottom:1px solid #996;"| 4
-
|style="border-bottom:1px solid #000;"| 5
+
|style="border-bottom:1px solid #996;"| 5
-
|style="border-bottom:1px solid #000;"| 6
+
|style="border-bottom:1px solid #996;"| 6
|-
|-
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|style="border-right:1px solid #000;"| Parts
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|style="border-right:1px solid #996;"| Parts
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| 1-18F
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]
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| 2-21H
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-21H]]
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| 2-11P
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-11P]]
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| 2-24G
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]
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| 1-2M
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]
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| 3-1E
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
|-
|-
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|style="border-right:1px solid #000;"| DNA solution
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|style="border-right:1px solid #996;"| DNA solution
| 2 uL
| 2 uL
| 2  
| 2  
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| 1  
| 1  
|-
|-
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|style="border-right:1px solid #000;"| 6x Sample Buffer
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|style="border-right:1px solid #996;"| 6x Sample Buffer
| 0.4 uL
| 0.4 uL
| 0.4  
| 0.4  
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| 0.4  
| 0.4  
|}
|}
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* マーカーはλ/''Hin''dIIIとpUC119/''Hin''fIを使用
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* Used markers are [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII] and [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''fI]
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* レーン1のマーカー(λ/''Hin''dIII)はウェルから流れ出てしまった
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* Marker in lane 1 ([https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII]) leaked out
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* レーン3(mini prepのサンプル,1-18F)はバンドが確認できなかった(後日PCR)
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* Mini prep sample,1-18F band in lane 3 wasn't visible so we [[Team:HokkaidoU_Japan/Protocols|decided]] PCR it tomorrow
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* レーン4ではplasmidのダイマー,トライマーがよく確認できる(plasmidの電気泳動ではよくあるらしい)
+
* In lane 4 there were some visible plasmid dimers and trimer s

Latest revision as of 07:25, 27 October 2010

Electrophoresis 1

Electrophoresis of yesterdays ligation of PCR products
  • Checked the result of yesterdays ligation of PCR products by electrophoresis
  • Added 4 uL of 6x Sample Buffer to make 12 uL in total
  • Used 20 uL of EtOH

Mini prep

  • Used cells incubated for 18h in LB broth
  • Centrifuge after adding 430 uL of chloroform、collected supernatant
  • Added 2-propanol to 1-18F, but after centrifugation there were no precipitation
    • Melted in 30 uL of TE and did electrophoresis

Electrophoresis 2

Take 1
Take 2 after few minutes

Checked plasmids gathered via mini prep and PCR products, by electrophoresis.


Because we wont do restriction enzyme digestion reaction system is ass follows.

Lane
Parts 1-18F 2-21H 2-11P 2-24G 1-2M 3-1E
DNA solution 2 uL 2 2 1 1 1
6x Sample Buffer 0.4 uL 0.4 0.4 0.4 0.4 0.4
  • Used markers are λ/HindIII and pUC119/HinfI
  • Marker in lane 1 (λ/HindIII) leaked out
  • Mini prep sample,1-18F band in lane 3 wasn't visible so we decided PCR it tomorrow
  • In lane 4 there were some visible plasmid dimers and trimer s