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 by annealing to the "barcode" added on step 1.
    • Reaction mix:
      • We do 2 different reactions one to a final volume of 25μl and one 50μl
      • In both cases the primers are BB5 (FW) and p230 (RV)
      • Same mix is used as in the first step, using the product as template.
    • PCR protocol:
      • Same protocol as step 1 but with annealing temperature of 60°C and only 10 cycles.
  3. The third step just amplifies the template with primers BB3 and BB5 (that anneal to prefix and suffix).
    • Reaction mix:
      • 25 final volume, 0.5 μl of each primer, rest as before.
    • PCR protocol:
      • Same protocol as step 1 but with 35 cycles.

The advantage of the 2-step PCR is that we only have to order the p230 primer once, at it is longer than 100bp and thus expensive. We can add Terminator and Suffix to any part by designing just a short annealing sequence with the "barcode" as 5' overhang.

Agarose gel: Results of the 2-Step PCR

Results:

The gel was loaded as follows:

  1. 1kb ladder
  2. 0.5μl template (50μl final volume)
  3. 2μl template (50μl final volume)
  4. 5μl template (50μl final volume)
  5. 0.5μl template (25μl final volume)
  6. 2μl template (25μl final volume)
  7. 5μl template (25μl final volume)
  8. 1kb ladder

The "best" band is on lane 3 (step 1 50μl, step 3 2μl). We digest it with X, P, then use PCR purification kit.

Simultaneously we digest R0071 with X, P. We want to cut out the part and use the vector to put our PCR'ed part in.

We ligate, transform and plate.

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