NYMU Taipei/Lab Notes/2007 8 6&7

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< NYMU Taipei/Lab Notes(Difference between revisions)
 
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<h3>Lab schedule</h3>
<h3>Lab schedule</h3>
-
* mouse insulin gene cloning (delay to 8/7)
+
* mouse insulin gene cloning (8/6 to 8/7)
** primer design for [http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI%3A96573 insulin II of Mus musculus], [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=142350788&dopt=GenBank&WebEnv=04iJq7mV5tF1N_LSoJWAy8uc-0i_vXvfkGc3KMxcxnrIPG9K-LDVOuYkZWjzh6BpujGcDGNGQk0k9I%40D4770B146B72B990_0041SID&WebEnvRq=1 MN_008387.3 transcript]
** primer design for [http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI%3A96573 insulin II of Mus musculus], [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=142350788&dopt=GenBank&WebEnv=04iJq7mV5tF1N_LSoJWAy8uc-0i_vXvfkGc3KMxcxnrIPG9K-LDVOuYkZWjzh6BpujGcDGNGQk0k9I%40D4770B146B72B990_0041SID&WebEnvRq=1 MN_008387.3 transcript]
 +
*** sequence for <font color="green">standard assembly (marked in green)</font> and <font color="red">PCR reaction (marked in red)</font>
*** primer for A chain
*** primer for A chain
**** translation (90-110): GIVDQCCTSICSLYQLENYCN
**** translation (90-110): GIVDQCCTSICSLYQLENYCN
**** transcript (341-403): GGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC, 63 bp
**** transcript (341-403): GGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC, 63 bp
-
**** left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATG<font color="red">ATTGTAGATCAGTGCTGCAC</font>
+
**** left: <font color="green">GTTTCTTCGAATTCGCGGCCGCTTCTAGATG</font><font color="red">GGCATTGTAGATCAGTGCTG</font>
-
**** right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA<font color="red">GTTGCAGTAGTTCTCCAGCT</font>
+
**** right: <font color="green">GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA</font><font color="red">GTTGCAGTAGTTCTCCAGCT</font>
 +
****Potential dimer formation! Do NOT use this pair of primers! We need a new design.
*** primer for B chain
*** primer for B chain
**** translation (25-54): FVKQHLCGSHLVEALYLVCGERGFFYTPMS
**** translation (25-54): FVKQHLCGSHLVEALYLVCGERGFFYTPMS
**** transcript (146-235):TTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCC, 90 bp
**** transcript (146-235):TTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCC, 90 bp
-
**** left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATG<font color="red">TTTGTCAAGCAGCACCTTTG</font>
+
**** left: <font color="green">GTTTCTTCGAATTCGCGGCCGCTTCTAGATG</font><font color="red">TTTGTCAAGCAGCACCTTTGT</font>
-
**** right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA<font color="red">GGACATGGGTGTGTAGAAGA</font>
+
**** right: <font color="green">GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA</font><font color="red">GGACATGGGTGTGTAGAAGA</font>
* purchase of BCRC human insulin clone (ordered not paid)
* purchase of BCRC human insulin clone (ordered not paid)
-
* cutting sites of parts
+
* cutting sites of iGEM 2007 parts
** ompR binding ompC promoter
** ompR binding ompC promoter
-
** tetR-EnvZ fusion construct
+
*** ompR binding site (C1, C2, C3) extraction
 +
*** assembly to insulin
 +
** [http://partsregistry.org/Part:BBa_C0082 tar-EnvZ fusion construct]: cut EnvZ part for our fusion construct
* antibiotics LB plate preparation
* antibiotics LB plate preparation
** ampicillin
** ampicillin
** kanamycin
** kanamycin
 +
[[Image:0806Plates.jpg|300px]]

Latest revision as of 09:18, 17 August 2007

Lab schedule

  • mouse insulin gene cloning (8/6 to 8/7)
    • primer design for [http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI%3A96573 insulin II of Mus musculus], [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=142350788&dopt=GenBank&WebEnv=04iJq7mV5tF1N_LSoJWAy8uc-0i_vXvfkGc3KMxcxnrIPG9K-LDVOuYkZWjzh6BpujGcDGNGQk0k9I%40D4770B146B72B990_0041SID&WebEnvRq=1 MN_008387.3 transcript]
      • sequence for standard assembly (marked in green) and PCR reaction (marked in red)
      • primer for A chain
        • translation (90-110): GIVDQCCTSICSLYQLENYCN
        • transcript (341-403): GGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC, 63 bp
        • left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGGCATTGTAGATCAGTGCTG
        • right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGTTGCAGTAGTTCTCCAGCT
        • Potential dimer formation! Do NOT use this pair of primers! We need a new design.
      • primer for B chain
        • translation (25-54): FVKQHLCGSHLVEALYLVCGERGFFYTPMS
        • transcript (146-235):TTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCC, 90 bp
        • left: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTTTGTCAAGCAGCACCTTTGT
        • right: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGGACATGGGTGTGTAGAAGA
  • purchase of BCRC human insulin clone (ordered not paid)
  • cutting sites of iGEM 2007 parts
    • ompR binding ompC promoter
      • ompR binding site (C1, C2, C3) extraction
      • assembly to insulin
    • [http://partsregistry.org/Part:BBa_C0082 tar-EnvZ fusion construct]: cut EnvZ part for our fusion construct
  • antibiotics LB plate preparation
    • ampicillin
    • kanamycin

0806Plates.jpg

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