Glasgow/Wetlab/Week1

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Contents

Week 1

Tuesday 3rd July 2007

  1. Maija and Christine prepared LB broth and LB agar with Protocol 1. Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.

Wednesday 4th July 2007

  1. All wetlab researched BioBricks.
  2. Reporter constructs and mini-Tn5 stocks looked out.
  3. Streaked the following:
    • P. putida PAW 340 pJAK14 (Carb. plate)
    • Tn5 lux AB (Carb. plate)
    • Mini-Tn5 lux AB (Carb. plate)
    • P. fluorescens NCIMB 9815 (Carb. plate)
    • P. putida KT2440 (LB)
    • JM109 pBluescript 5k+ (Carb. plate)
    • Mini-Tn5 Tc (Carb. Plate)
    • pQF52 (Carb. plate)
    • P. fluorescens 9815 (LB)
    • E. coli pJAK14 (Km plate)
    • Il DntR in pOF52 (Carb. plate)
    • pUCINR in Ω strain C (Carb. plate)
    • pGLTUR (Carb. plate)
    • Mini-Tn5 Kan (Carb. plate)
    • Mini-Tn5 Sm/Sp (Carb. plate)
    • Mini-Tn5 1cc2 (Carb. plate)
    • E. coli Sa1 (LB)
    • DmpR #24 (Carb. plate)
    • Mini-Tn5 lac 32 in E. coli 517 (Carb. plate)
    • Mini-Tn5 Tc (Carb. Plate)
    • Mini-Tn5 Cm (Carb. plate)
    • DmpR WT (Carb. plate)
    • E.coli sm 10 pESD15 Tn5 GFP (Carb. plate)
    • pUJ8 (Carb. Plate)

Thursday 5th July 2007

  1. BioBricks – Maija and Scott transformed using Protocol 2.
    BioBrick Part Function Chassis Plasmid Kit Plate Location (Plate/Well)
    [http://partsregistry.org/Part:BBa_P1010 Bba_p1010] Construction plasmid E. coli DB3.1 pSB1A10 4/7A
    " " " pSB1A10 4/11E
    " " " pSB3K3 4/11C
    [http://partsregistry.org/Part:BBa_I52001 Bba_I52001] High copy-number construction plasmid E. coli TOP10
    should have been DB3.1
    pSB4A5 4/5I
    " " " pSB4K5 4/5D
    " " " pSB4K5 4/6D
    " " " pSB3K5 4/6B
    " " " pSB4A5 4/6E
    [http://partsregistry.org/Part:BBa_J23119 Bba_J23119] Strong constitutive promoter E. coli TOP10 pSB1A2 3/19A
    [http://partsregistry.org/Part:BBa_R0062 Bba_R0062] HSL & LuxR-inducible promoter E. coli TOP10 pSB1A2 1/9G
    [http://partsregistry.org/Part:BBa_J04500 Bba_J04500] IPTG-inducible promoter + RBS E. coli TOP10 pSB1AK3 1/16P
    [http://partsregistry.org/Part:BBa_E0040 Bba_E0040] Promoter-less GFP E. coli TOP10 pSB1A2 1/5H
  2. Maia and Christine researched restriction enzymes for the reporter constructs and Mini-Tn5s streaked on 4/7/07 and digested some. Had planned to make biobricks using Mini-Tn5s etc but discovered that [http://partsregistry.org/Part:BBa_J61206 a previous attempt to use transposable elements to make biobricks] had been unsuccessful due to scarring at the restriction site.

Friday 6th July 2007

  1. Maija and Christine made 10x stocks for M9 (see Protocol 3.2 - M9).
  2. Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
  3. Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see Protocol 4).


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