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To do today

  • Grow up 12D->2H for miniprep this afternoon
  • Miniprep 12D->2H
  • Pick up 2 bottles of LB (no Amp) from cold room in BCHM
  • Stage 3 Ligations
    • J37016 = J37019 (7A->3O->9G) + I13504 (12D)
    • J37015RS = F2620 (6B) + (RS+C0261+I13504) (12D->24A->RS)


  • The ligated part "A" - 12D->24A->6B (J37015) was maxipreped.
  • Digested with EcoR1 and Pst1 and run on a gel
  • The digest should leave 2 bands - one for the plasmid and one for the 3 parts ligated together
  • Two very clear bands were found (the part was run twice in seperate lanes)
  • The gel image can be seen below.

J37015 gel.GIF

  • The bands are at around 2kb and 2.5kb, which is as expected
  • Success!! We have our first construct!!!


  • As part "A" (J37015) is the final prey assembly, we plated up the bacteria with this plasmid.


Immunotag Fusion PCR carried out, using recently arrived new oligos. Left to run overnight. Will run on gel tomorrow and isolate DNA using glass milk preparation. If successful, will start Stage 1 ligation of the AiiA construct. 3rd time lucky???


  • Ligate the following
    • J37016 = J37019 (vector) + I13504 (insert)
      • J37019 used colonies R, V, Z
    • J37015 = F2620 (vector) + (RS+C0261+I13504) (insert)
      • (RS+C0261+I13504) used colonies M & L
  • Electroporation to do tomorrow

To do tomorrow

  • Miniprep of ligations from Tuesday (colonies are in shaker)
  • Electorporation of Ligations from today
  • Sequencing DNA to Dr. Mann for part J37015