Team:Todai-Tokyo/Protocols/Notebook Sample

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Miniprep

The following were miniprepped:

• pLacI-RFP-dterm (From Ligation on 9/4): 4 colonies
• RFP (BBa_J04450 from 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 (BBa_J04451 from 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
  • pLacI-RBS S/P (Digested on 10/1)
  • yqiT-dterm E/X (Digested on 10/1)
• pAraC-RBS-yqiT-dterm
  • pAraC-RBS S/P (Digested on 9/22)
  • yqiT-dterm E/X (Digested on 10/1)

Infusion + Transformation

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

• pLacI-RBS-yqiT-dterm
  • pLacI-RBS S/P (Digested on 10/1)　as vector
  • yqiT-dterm (Miniprepped on 10/1) as insert

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:

• pLacI (Miniprepped on 10/1): SpeI/PstI
• RFP-dterm (Miniprepped on 10/1): XbaI/PstI

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