Team:TU-Delft/Protocol 5

From 2013.igem.org

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<center>Table 1: Lysis experiment </center>
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                         <td> Component </td>
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Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.
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<ol>Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.</ol>
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Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.  
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<ol> Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly. </ol>
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Latest revision as of 15:27, 3 October 2013



Protocols

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.



Restriction digestion

Requirements:

  1. plasmid DNA or PCR product
  2. restriction enzymes (Roche and BioLabs)
  3. buffer (10x)
  4. H2O
  5. water bath at 37 °C
  6. heat block or water bath at 65 °C

Procedure:

  1. Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
  2. Reaction for one sample:
    3. Component Sample
    DNA x μL (up to 1,0 μg)
    Buffer (10x) x μL (for 1×)
    Restriction enzymes x μL (10 units/μg DNA = 1 µL)
    H2O x μL
    20-25 μL

    Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.

    Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.

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