EPF-Lausanne/29 September 2008

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Cloning

2-step PCR of LuxI

The goal is to obtain the following part: BBprefix-RBS-LuxI-Terminator-BBsuffix

This is done by 2-step PCR:

  1. A first set of primers adds a "barcode"-sequence to the LuxI which is present on a the plasmid pSND (from R.Weiss).
    • Reaction mix:
      • 2.5 μl Buffer 10X
      • 0.75 μl MgCl2
      • 0.5 μl dNTP
      • 0.5 μl pSND1 (template)
      • 0.5 μl p151 (FW primer)
      • 0.5 μl p131 (RV primer)
      • 0.1 μl TAQ
      • 14.65 μl Water
    • PCR protocol:
      1. 94°C, 4 minutes
      2. 94°C, 30 seconds
      3. 55°C, 1 minute
      4. 72°C, 2 minutes
      5. cycle 2-4 30x
      6. 72°C, 5 minutes
      7. 4°C, inf.
  2. A second set of primers adds RBS (B0034) and Terminator (B0015) together with the prefix and suffix.
    • Reaction mix:
      • We do 2 different reactions and each one to a final volume of both 25 μl and 50 μl
      1. Same mix is used as in the first step, using the product as template.
      2. Here the primer volumes are 5 μl each, otherwise same as step 1.

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