PCR – Withering Syndrome Bacteriophage Clone Screen

10 colonies from both ligations (pCR2.1/ORF20 & pCR2.1/ORF25) were picked with clean pipette tips, streaked on a different LB-Amp50+X-gal plate that had been gridded and numbered, and then used to inoculate the PCR reactions.

PCRs were performed with respective phage primers.

Master mix calcs are here: 20140909 – Phage Colony Screens

Cycling params:

1 cycle:

  • 95C – 10mins

40 cycles:

  • 95C – 15s
  • 55C – 15s
  • 72C – 30s

Results:

Top half of gel are the pCR2.1/ORF20 colonies.

Bottom half of gel are the pCR2.1/ORF25 colonies.

All amplified. Will select one of each from the gridded plates for plasmid isolation for use in qPCR standard curves and in-situ hybridization (ISH).

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