Team:Todai-Tokyo/Protocols/Notebook Sample

From 2009.igem.org

(Difference between revisions)
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|style="width:15%;"|
|style="width:15%;"|
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<html></html>
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<html>
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<font size = 4><B>このページの使い方</b></font><BR><BR>
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各メソッドのサンプルです。<BR>
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ソース(edit画面）からそれぞれをコピーして使いたいところに貼り付け、必要なところを変える。<BR>
 +
＊このページに登場する作業やDNA配列はフィクションです。<BR><BR>
 +
 
 +
 
 +
</html>
<h2> Miniprep </h2>
<h2> Miniprep </h2>
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The following were [[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped:
The following were [[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped:
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* pLacI-RFP-dterm (From Ligation on 9/4; 4 colonies)
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* pLacI-RFP-dterm (From Ligation on 9/4): 4 colonies
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* RFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution]; 6 colonies)
+
* RFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution]): 6 colonies
 +
 
<h2> PCR </h2>
<h2> PCR </h2>
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Note: This PCR reaction attaches a Biobrick prefix and suffix to the gene.
Note: This PCR reaction attaches a Biobrick prefix and suffix to the gene.
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 +
＊補足： Program 1と書いてあるところは、いつも使うようなプログラムと違うものを使ってそれを記したい場合はプログラムの内容をリンク先に書き込みましょう。わからない場合は聞いてください。
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* pAraC Sample 2 ([[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped on 9/23)
* pAraC Sample 2 ([[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped on 9/23)
** 3) '''Biobrick vector fwd''': atatgagagcgcgcgcagagagagatg
** 3) '''Biobrick vector fwd''': atatgagagcgcgcgcagagagagatg
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<BR>
'''Results''':<BR>
'''Results''':<BR>
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<BR>2) Single-base mutation found in sequence that creates stop codon; re-do the ligation
<BR>2) Single-base mutation found in sequence that creates stop codon; re-do the ligation
<BR>3) Desired Sequence read
<BR>3) Desired Sequence read
 +
<h2> Transformation </h2>
<h2> Transformation </h2>
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* pAraC-RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04451 BBa_J04451] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution])
* pAraC-RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04451 BBa_J04451] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution])
* pLacI ([[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped on 9/23)
* pLacI ([[Team:Todai-Tokyo/Protocols/Miniprep|miniprep]]ped on 9/23)
 +
<h2> Ligation + Transformation </h2>
<h2> Ligation + Transformation </h2>
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** pLacI-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
** pLacI-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
** yqiT-dterm '''E/X''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
** yqiT-dterm '''E/X''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
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*pAraC-RBS-yqiT-dterm
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* pAraC-RBS-yqiT-dterm
** pAraC-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 9/22)
** pAraC-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 9/22)
** yqiT-dterm '''E/X''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
** yqiT-dterm '''E/X''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
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<h2> Infusion </h2>
<h2> Infusion </h2>
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<h2> Gel Purification </h2>
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<h2> RE Digest </h2>
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＊ Infusionを理解してないので間違ってるかもしれません。誰かこの手法を教えてください。
 +
 
 +
The following constructs were created using [[Team:Todai-Tokyo/Protocols/Infusion|Infusion]] and the listed fragments:
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* pLacI-RBS-yqiT-dterm
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** pLacI-RBS ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1)
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** yqiT-dterm  ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1)
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 +
 
 +
<h2> RE Digest + Gel Purification </h2>
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 +
The following were [[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] using the listed Restriction Enzymes and then [[Team:Todai-Tokyo/Protocols/Gel Purification|Gel Purified]]:
 +
 
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* pLacI ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1): '''SpeI/PstI'''
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* RFP-dterm ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1): '''XbaI/PstI'''
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<h2> Colony PCR </h2>
<h2> Colony PCR </h2>
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Colonies from the following plates were used for [[Team:Todai-Tokyo/Protocols/Colony PCR|Colony PCR]] using the listed primers:
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* EGFP (Transformed on 9/21): 4 colonies
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**'''EGFP rev''': atatgagagcgcgcgcagagagagatg
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* Venus (Transformed on 9/21): 4 colonies
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**'''Venus fwd''': atatgagagcgcgcgggggagagagagatg

Revision as of 04:05, 20 October 2009

UT.png
Home The Team The Project Parts Submitted to the Registry Modeling Notebook Protocols Ethics



このページの使い方

各メソッドのサンプルです。
ソース(edit画面）からそれぞれをコピーして使いたいところに貼り付け、必要なところを変える。
＊このページに登場する作業やDNA配列はフィクションです。

Contents

Miniprep

The following were miniprepped:

  • pLacI-RFP-dterm (From Ligation on 9/4): 4 colonies
  • RFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution]): 6 colonies


PCR

The yqiT gene was PCR amplified from pLacI-yqiT-dterm (miniprepped on 9/7) using the following primers:

yqiT fwd: agcccgtgtagtactgtagagtt
yqiT rev: agggatgtttgccagtcgtaattag

using PCR program 1 and Ex-taq.

Note: This PCR reaction attaches a Biobrick prefix and suffix to the gene.

＊補足： Program 1と書いてあるところは、いつも使うようなプログラムと違うものを使ってそれを記したい場合はプログラムの内容をリンク先に書き込みましょう。わからない場合は聞いてください。


Sequencing

The following were sequenced using the labeled primers:

  • pAraC Sample 1 (miniprepped on 9/23)
    • 1) Biobrick vector fwd: atatgagagcgcgcgcagagagagatg
    • 2) Biobrick vector rev: tttttaaaagggggtgtgtgtgtaaagttt
  • pAraC Sample 2 (miniprepped on 9/23)
    • 3) Biobrick vector fwd: atatgagagcgcgcgcagagagagatg


Results:
1) Sequence read failed
2) Single-base mutation found in sequence that creates stop codon; re-do the ligation
3) Desired Sequence read


Transformation

The following were Transformed into E. coli competent cells:

  • pAraC-RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J04451 BBa_J04451] from [http://partsregistry.org/assembly/libraries.cgi?id=19 2009 Spring Distribution])
  • pLacI (miniprepped on 9/23)


Ligation + Transformation

The following Ligations were performed using the listed fragments and Transformed into E. coli competent cells:

  • pLacI-RBS-yqiT-dterm
  • pAraC-RBS-yqiT-dterm


Infusion

＊ Infusionを理解してないので間違ってるかもしれません。誰かこの手法を教えてください。

The following constructs were created using Infusion and the listed fragments:


RE Digest + Gel Purification

The following were Digested using the listed Restriction Enzymes and then Gel Purified:


Colony PCR

Colonies from the following plates were used for Colony PCR using the listed primers:

  • EGFP (Transformed on 9/21): 4 colonies
    • EGFP rev: atatgagagcgcgcgcagagagagatg
  • Venus (Transformed on 9/21): 4 colonies
    • Venus fwd: atatgagagcgcgcgggggagagagagatg
Retrieved from "http://2009.igem.org/Team:Todai-Tokyo/Protocols/Notebook_Sample"