Team:Todai-Tokyo/Protocols/Notebook Sample

From 2009.igem.org

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<BR>
<BR>
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'''Results''':<BR>
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'''Results''':<BR><BR>
1) Sequence read failed
1) Sequence read failed
<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
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<h2> Infusion + Transformation </h2>
<h2> Infusion + Transformation </h2>
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The following constructs were created using [[Team:Todai-Tokyo/Protocols/Infusion|Infusion]] and the listed fragments:
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The following constructs were created using [[Team:Todai-Tokyo/Protocols/Infusion|Infusion]] and the listed fragments, then [[Team:Todai-Tokyo/Protocols/Transformation|Transformed]] into ''E. coli'' competent cells:
* pLacI-RBS-yqiT-dterm
* pLacI-RBS-yqiT-dterm
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** pLacI-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1)
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** pLacI-RBS '''S/P''' ([[Team:Todai-Tokyo/Protocols/Restriction Enzyme Digest|Digested]] on 10/1) as vector
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** yqiT-dterm  ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1)
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** yqiT-dterm  ([[Team:Todai-Tokyo/Protocols/Miniprep|Miniprepped]] on 10/1) as insert
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using the following primers:<BR>
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* '''Inf fwd''': agtgtgtcgatgcagcccatgacacacacacagagatag
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* '''Inf rev''': ggggagagatatatagagagacacacacagagatatat

Latest revision as of 10:14, 20 October 2009

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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 + Transformation

The following constructs were created using Infusion and the listed fragments, then Transformed into E. coli competent cells:

  • pLacI-RBS-yqiT-dterm

using the following primers:

  • Inf fwd: agtgtgtcgatgcagcccatgacacacacacagagatag
  • Inf rev: ggggagagatatatagagagacacacacagagatatat


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