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Methods:single lambda lysogen confirmation by PCR

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Contents

Purpose

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Materials Needed

Protocol

From Powell et al. (1994)[1]

To prep colony for PCR:

  1. Restreak lysogenized colonies & incubate overnight at appropriate temperature.
  2. Pick a single colony into 500μL sterile water. Resuspend.
  3. Spin at 15 000 rpm for 2 mins.
  4. Aspirate off supernatant IMMEDIATELY and CAREFULLY!
  5. Resuspend in 500μL sterile water. Repeat steps 3-5 twice.
  6. Resuspend the pellet in 100μL sterile water.
  7. Use 20μL of the cell suspension in a PCR.

PCR:

    • cell suspension 20μL
    • Primer mix (10μM of each primer of each of the three primers listed in the reference) 2.5μL
    • Taq Buffer 5μL
    • 10mM dNTP mix 1μL
    • Taq 0.5μL
    • water 21.5μL
  • total 50μL

PCR conditions:

  • begins with:
    • 95oC - 1 min
  • repeat 25X:
    • 95oC - 1 min
    • 55oC - 1 min
    • 72oC - 1 min
  • ends with:
    • 72oC - 5 min
  • run 10μL of PCR on 1.5% agarose gel
single lysogen = 501 bp band
multiple lysogen = 501 bp & 379 bp bands 

Usage Examples

User notes

Related GO Terms

References

  1. Powell, BS et al. (1994) Rapid confirmation of single copy lambda prophage integration by PCR. Nucleic Acids Res. 22 5765-6 PubMed EcoliWiki page