Team:Calgary/1 July 2009

• High Arctic plant rhizosphere(P. putida)
• Mid-gut of frozen beetle larvae (R. erythropolis)
• Frozen/chilled pork sausages (M. cryophilus)
• Antarctic soil (Moraxella sp and P. fluorescens).

None were from bacteria until MpAFP was found

In the lab Shadowed Vicky with Colony PCR of J13002 + JuxOD47A BBk + B0015 on pSB1AK3 Purpose: The bacterial culture growth is prolific. This will verify if the proper gene constructs are included within.

Materials an methods:

• Primers: BBk forward and reverse construction primers
• Master mix: Use pTaq DNA polymerase. The contents will suffice for 15 tubes, and were prepared in the same fashion as on June 26.
• PCR conditions: These are also the same as they were on June 26

Tubes 1/6 were colony 1

tubes 2/7 were col 2

tubes 3/8 were col 3

tubes 4/9 were col 4

tubes 5/10 were col 5

Materials an methods:

• Primers: BBk forward and reverse construction primers
• Master mix: Use pTaq DNA polymerase. The contents will suffice for 15 tubes, and were prepared in the same fashion as on June 26.
• PCR conditions: These are also the same as they were on June 26

Results:

The 1% agarose gel is attached below. Lane 1 is a GeneElute 1kb+ DNA ladder; lanes 2-11 are the colony-PCR'd samples of J13002 + LuxOD47A + B0015; lane 12 is a sequenced LuxOD47A + B0015 for a size control; lane 13 is a sequenced LuxOD47A BBk for another size control; and lane 14 is a negative control. Based on a very confusing negative control, this procedure will be repeated tomorrow. We will still progress with a NotI restriction digest of the contents, which will also be done tomorrow.