Team:Warsaw/Calendar-Main/28 August 2009

From 2009.igem.org

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<li>After seting electrophoresis I found out that the digestion was preparet wrong, also gel went wrong. </li>
<li>After seting electrophoresis I found out that the digestion was preparet wrong, also gel went wrong. </li>
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<h3><div style="text-align: center;">Cloning switch 1 regulatory parts [
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI,
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator,
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator,
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<a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator
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]
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into two compatible low copy number plasmids of different antibiotic resistance</div></h3>
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<h4>Ania</h4>
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<p>Tasks:</p>
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<ul>
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<li>
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</li>
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</ul>
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<br/>
</html>
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Revision as of 00:19, 10 September 2009


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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Amplification of the mgtc promoter using new primers
  • pSB1A3 plasmid digest

Methods:

  • The PCR mix was prepared as follows:

    5μl buffer B (EURx)
    2μl 5mM dNTPs (EURx)
    5μl forward starter
    5μl reverse starter
    2μl OptiTaq polymerase (EURx)
    2μl Salmonella matrix
    29 μl H2O
  • PCR programme:

     4min 95°C 
    (30s 95°C, 35s 48°C, 40s 72°C)x28
    (30s 95°C, 35s 58°C, 40s 72°C)x28
    10min 72°C
    ~ 4°C
  • The PCR results were visualised with gel electrophoresis on 1% agarose gel.
  • The digest mix was prepared as follows:

    60μl plasmid isolation
    7μl Buffer O (Fermentas)
    1μl EcoRI enzyme
    1μl PstI enzyme
    1μl H2O
  • The digest was carried out in 37°C for 3h and inactivated for 15 min. in 80°C.
  • 1μl of CIAP enzyme was added after the inactivation and allowed to work for 1h in 37°C and was later inactivated for 15 min. in 85°C.
  • The resulting mix was separated on 1% agarose gel alongside the PCR and then selected bands were extracted for purification.

Results:

  • No gel picture, but the PCR worked so great that I decided not to irradiate the gel with UV light more than it was necessary for band extraction.

Conclusions:

  • The temperatures were chosen... wisely.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Cro-box digest

Methods:

  • The digest mix was prepared as follows:

    20μl plasmid isolation
    3μl Buffer O (Fermentas)
    1μl EcoRI enzyme
    1μl PstI enzyme
    5μl H2O

Assembly of endosomal detection operon

Marcin


Task 1:

Methods:

  • All gel-outs were performed using the EurX gel-out kit according to the manual

Results:

  • Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation

Comment:

Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid was insufficient to perform gel-out. It is obligatory to digest this construct one more time


Task 2:

  • Digestion of following construct:
  • BBa_C0051 with BBa_B0032 on pSB1A3 plasmid
  • Methods:

    • Reaction mixture composition:
    •  
      20 μl purified plasmid DNA product
      1 μl XbaI (Fermentas)
      1 μl PstI (Fermentas) 
      2 μl Buffer Tango (Fermentas)
      15 μl MQ water
    • Digest was performed about six hours and subsequently thermal inactivated

    Task 3:

    • Gel-outs of construct described in Task 2

    Methods:

    • All gel-outs were performed using the EurX gel-out kit according to the manual

    Results:

    • Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation

    Comment:

    Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid may be insufficient for efficient gel-out. Although I decided to continue the task


    Task 4:


    Methods:

    • Ligation mixtures composition:
    •  
      30 μl digested insert
      10 μl digested vector 
      5 μl Tango buffer(Fermentas)
      3 μl dNTPs mixture (EurX, concentration 5 mM) 
      2 μl ligase T4 (Fermentas)
    • Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).
    • In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes

    Making of the plac-RBS-llo-intA part

    Jarek


    Tasks:

    • Digestion of pUC19-intA with PstI and CaiI endonucleases, and plac-RBS-llo with PstI (this sample was additionaly defosforylated during digestion).
    • Separation of digested fragments in 0,8% agarose gel
    • Another digestion of pUC19-intA and plac-RBS-llo with the same enzymes

    Results: