Team:UNIPV-Pavia/Notebook/Week3

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Revision as of 13:22, 7 June 2008


Home.jpg Home Unipv logo.jpg The Team And.jpg The Project Safety.jpg Biological Safety Dna.png Parts Submitted to the Registry
Laptop.jpg Dry Lab Pipette.jpg Wet Lab Math.gif Modeling Note.jpg Protocols Notebook.gif Activity Notebook



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) and BBa_B0030 (X-P)
    • one for BBa_C0061 (X-P), BBa_I15008 (X-P) and BBa_I15010 (X-P).
  • We could see and cut all the bands we wanted, except for BBa_I14032, for which we couldn't see any band.
  • We performed gel extraction for:
BBa_R0051 (S-P) BBa_B0030 (X-P) BBa_C0061 (X-P) BBa_I15008 (X-P)
BBa_I15010 (X-P)
Mattia cutting BBa_C0061 (X-P), BBa_I15008 (X-P) and BBa_I15010 (X-P) gel
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