Team:UNIPV-Pavia/Notebook/Week3

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Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7



Week 3: 06/03/08 - 06/06/08

06/03/08

  • We transformed 60 µl of TOP10 with 2 µl of the 6 parts (DNA + glycerol) we received from IGEM HQ:
BBa_C0179 BBa_C0161 BBa_R0051 BBa_I14032
BBa_I15008 BBa_I15010
  • NOTE: We noticed that IGEM 2007 teams which used luxI or lasR parts choosed BBa_C0061 instead of BBa_C0161 and BBa_C0079 instead of BBa_C0179. So, we decided in addition to amplify BBa_C0061 and BBa_C0079; we shall see later which luxI and lasR to choose.
  • So, we cut paper spots for BBa_C0061 and BBa_C0079 and resuspended them in 10 µl of warmed TE buffer.
  • We transformed these 2 parts using 4 µl of DNA in TE.
  • We plated transformed bacteria and incubated them at 37°C overnight.



06/04/08

  • After overnight incubation, the following 5 plates showed colonies:
BBa_C0061 BBa_R0051
BBa_I14032 BBa_I15008
BBa_I15010
BBa_R0051 plate: very high yield from IGEM HQ DNA + glycerol
BBa_C0061 plate: medium yield from paper spot
  • while the following 3 plates did not:
BBa_C0079 BBa_C0179
BBa_C0161
  • We repeated the transformation for BBa_C0079, BBa_C0179 and C0161. We used 6 µl of DNA in TE for BBa_C0179, while we used 3 µl of DNA + glycerol for BBa_C0079 and BBa_C0161.
  • We plated transformed bacteria and incubated them at 37°C overnight.
  • While we were preparing our 5 ml cultures, we noticed that LB + Amp was contaminated! We decided to prepare a big quantity of LB + Amp and also of LB + Kan: we prepared 0.5 l LB + Amp and 0.5 l LB + Kan for liquid cultures; 0.5 l LB + Amp and 0.5 l LB + Kan for plates.
LB plates we just prepared
  • We picked up one colony from BBa_C0061, BBa_R0051, BBa_I14032, I15008 and I15010 plates to grow 5 ml cultures of transformed bacteria overnight.
  • We also infected 5 ml of LB + Amp with 15 µl of BBa_B0030 glycerol stock. We incubated the 5 ml cultures overnight at 37°C.
  • We received QIAGEN QIAprep Spin Miniprep Kit!!! We will inaugurate it tomorrow on these 5 ml cultures;)



06/05/08

  • We received Euroclone Antartic Phosphatase: we will use it in the afternoon!
  • We prepared 14 glycerol stocks taking 800 µl from 5 ml cultures containing:
BBa_R0051 BBa_I15008 BBa_I15010 BBa_I14032
BBa_C0061 BBa_B0030
  • We performed miniprep for these 6 parts with our new splendid kit;) Plasmid quantification confirmed a higher yield than our previous QIAGEN kit.
  • We performed plasmid digestion for these parts (20 of 30 µl).
  • We had to insulate excided fragments for:
BBa_I15008 (X-P) BBa_I15010 (X-P) BBa_I14032 (X-P) BBa_C0061 (X-P)
BBa_B0030 (X-P)
  • While we had to insulate opened plasmids for:
BBa_R0051 (S-P)
  • Due to the different dimension of the DNA we had to insulate, we ran 3 different gels:
    • one for BBa_R0051 (S-P)
    • one for BBa_I14032 (X-P)
    • one for BBa_C0061 (X-P), BBa_I15008 (X-P), BBa_I15010 (X-P), BBa_B0030 (X-P).
  • Unfortunately, BBa_B0030 (X-P) and BBa_J23100 (E-S) fragments had smearing appearance because they were smaller than other excided fragments and ran too fast.
  • We performed gel extraction for all the 9 parts, but we decided to repeat cutting/run/extraction process for BBa_B0030 (X-P) and BBa_J23100 (E-S) in the next days, to perform a more efficient extraction for these 2 parts.