Glasgow/Wetlab/Week1

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[[Glasgow|Glasgow Main Page]] | [[Glasgow/Wetlab|Back To Wetlab Log]]
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!align=center|[[Image:Uog.jpg]] ||  [[Glasgow|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Main Page</font>]] || [[Glasgow/Wetlab|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Wetlab Log</font>]] ||  [[Glasgow/Modeling|<font face=georgia color=#3366CC size=4>Go To <br> Glasgow's <br>Drylab Log</font>]]
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----
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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|- align=center
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|[https://2007.igem.org/Glasgow/Wetlab/Protocols <font face=georgia color=#3366CC size=5><b>Protocols</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/References <font face=georgia color=#3366CC size=5><b>References</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Resources <font face=georgia color=#3366CC size=5><b>Resources</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Orders <font face=georgia color=#3366CC size=5><b>Orders</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Biobricks <font face=georgia color=#3366CC size=5><b>Biobricks<br>Used</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Gels <font face=georgia color=#3366CC size=5><b>Gels</b></font>]
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|}
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== Week 1 ==
== Week 1 ==
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=== Tuesday 3 July 2007===
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=== Tuesday 3rd July 2007===
#[[User:MaijaP|Maija]] and [[User:Christinemerrick|Christine]] prepared LB broth and LB agar with [[Glasgow/Wetlab/Protocols# Protocol 1: LB Broth and Agar|Protocol 1]].  Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.
#[[User:MaijaP|Maija]] and [[User:Christinemerrick|Christine]] prepared LB broth and LB agar with [[Glasgow/Wetlab/Protocols# Protocol 1: LB Broth and Agar|Protocol 1]].  Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.
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=== Wednesday 4 July 2007===
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=== Wednesday 4th July 2007===
#All wetlab researched BioBricks.
#All wetlab researched BioBricks.
#Reporter constructs and mini-Tn5 stocks looked out.
#Reporter constructs and mini-Tn5 stocks looked out.
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#*pUJ8 (Carb. Plate)
#*pUJ8 (Carb. Plate)
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=== Thursday 5 July 2007 ===
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=== Thursday 5th July 2007 ===
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#BioBricks – [[User:MaijaP|Maija]] and [[User:scott.w.ramsay|Scott]] transformed using [[Glasgow/Wetlab/Protocols#Protocol 2: Transforming Biobricks | Protocol 2]].
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<ol>
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#*BBa_p1010 (DB3.1) (death gene plasmid) Plate 4: 7A – p5B1A10, 11E – p5B1A10 and 11C – p5B3K3
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<li>BioBricks – [[User:MaijaP|Maija]] and [[User:scott.w.ramsay|Scott]] transformed using [[Glasgow/Wetlab/Protocols#Protocol 2: Transforming Biobricks | Protocol 2]].
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#*BBa_IS2001 (Top10) (high copy number plasmid) Plate 4: 5I – p5B4A5, 5D – p5B4K5, 6B – p5B3K5, 6D – p5B4K5 and 6E – p5B4A5 (Also 5K, 5M, 4J, 4L, 4N, 4P)
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#*BBa_J23119 (Top 10) (strong constitutive promoter) Plate 3: 19A – pB1A2 (V1013)
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#*BBa_R0062 (Top 10) (HSL and luxR inducible) Plate 1: 9G – pSB1A2 (V1004)
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#*BBa_J04500 (Top 10) (IPTG inducible prom + RBS) Plate 1: 16P – p5B1AK3 (V1009)
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#*BBa_E0040 (Top 10) (GFP mutant no promoter (3b)) Plate 1: 5H – p5B1A2 (V1001)
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#[[User:Mojs|Maia]] and [[User:Christinemerrick|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 a previous attempt to use transposable elements to make biobricks had been unsuccessful due to scarring at the restriction site. http://partsregistry.org/Part:BBa_J61206
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=== Friday 6 July 2007 ===
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{| align="center" border="1" cellpadding="5"
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|- align="center"
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|'''BioBrick Part''' || '''Function''' || '''Chassis''' || '''Plasmid''' || '''Kit Plate Location''' ''(Plate/Well)''
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|- align="center"
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|[http://partsregistry.org/Part:BBa_P1010 Bba_p1010] || Construction plasmid || ''E. coli''  DB3.1 || pSB1A10 || 4/7A
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|- align="center"
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| " || " || " || pSB1A10 || 4/11E
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|- align="center"
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| " || " || " || pSB3K3 || 4/11C
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|- align="center"
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|[http://partsregistry.org/Part:BBa_I52001 Bba_I52001] || High copy-number construction plasmid || ''E. coli''  TOP10 <br> <font color="red">should have been DB3.1</font> || pSB4A5 || 4/5I
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|- align="center"
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| " || " || " || pSB4K5 || 4/5D
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|- align="center"
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| " || " || " || pSB4K5 || 4/6D
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|- align="center"
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| " || " || " || pSB3K5 || 4/6B
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|- align="center"
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| " || " || " || pSB4A5 || 4/6E
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|- align="center"
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|[http://partsregistry.org/Part:BBa_J23119 Bba_J23119] || Strong constitutive promoter || ''E. coli''  TOP10 || pSB1A2 || 3/19A
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|- align="center"
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|[http://partsregistry.org/Part:BBa_R0062 Bba_R0062] || HSL & LuxR-inducible promoter || ''E. coli''  TOP10 || pSB1A2 || 1/9G
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|- align="center"
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|[http://partsregistry.org/Part:BBa_J04500 Bba_J04500] || IPTG-inducible promoter + RBS || ''E. coli''  TOP10 || pSB1AK3 || 1/16P
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|- align="center"
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|[http://partsregistry.org/Part:BBa_E0040 Bba_E0040] || Promoter-less GFP || ''E. coli''  TOP10 || pSB1A2 || 1/5H
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|}
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</li>
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<li>
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[[User:Mojs|Maia]] and [[User:Christinemerrick|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.
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</li>
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</ol>
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=== Friday 6th July 2007 ===
#[[User:MaijaP|Maija]] and [[User:Christinemerrick|Christine]] made 10x stocks for M9 (see [[Glasgow/Wetlab/Protocols#Protocol 3: Minimal Media and Trace Elements|Protocol 3.2 - M9]]).
#[[User:MaijaP|Maija]] and [[User:Christinemerrick|Christine]] made 10x stocks for M9 (see [[Glasgow/Wetlab/Protocols#Protocol 3: Minimal Media and Trace Elements|Protocol 3.2 - M9]]).
#Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
#Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
#Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see [[Glasgow/Wetlab/Protocols#Protocol 4: Primer Design|Protocol 4]]).
#Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see [[Glasgow/Wetlab/Protocols#Protocol 4: Primer Design|Protocol 4]]).
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<br>
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[[Glasgow/Wetlab/Week2|<font face=georgia color=#3366CC size=4>Next <br>  Week</font>]]
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----
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{| valign=top cellpadding=3
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!align=center|[[Image:Uog.jpg]] ||  [[Glasgow|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Main Page</font>]] || [[Glasgow/Wetlab|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Wetlab Log</font>]]
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|}

Latest revision as of 17:07, 22 October 2007

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Used 		Gels

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)
    Bba_p1010 Construction plasmid E. coli DB3.1 pSB1A10 4/7A
    " " " pSB1A10 4/11E
    " " " pSB3K3 4/11C
    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
    Bba_J23119 Strong constitutive promoter E. coli TOP10 pSB1A2 3/19A
    Bba_R0062 HSL & LuxR-inducible promoter E. coli TOP10 pSB1A2 1/9G
    Bba_J04500 IPTG-inducible promoter + RBS E. coli TOP10 pSB1AK3 1/16P
    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 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 BioEdit and Primer3.
  3. Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see Protocol 4).


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