Team:UNIPV-Pavia/Notebook/Week5

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Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14
Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21
Week 22 Week 23 Week 24



Week 5: 06/16/08 - 06/20/08

06/16/08

  • We picked up the only colony in BBa_B0030-BBa_C0078 plate to grow a 9 ml culture of transformed bacteria overnight.
  • We also infected 9 ml of LB + suitable antibiotic with 30 µl of:
BBa_B0030 BBa_E1010 BBa_E0040
BBa_C0061 BBa_C0051
  • glycerol stocks. We incubated the 9 ml culture overnight at 37°C, 220 rpm.



06/17/08

  • Glycerol stocks for:
BBa_B0030 BBa_E1010 BBa_E0040
BBa_C0061 BBa_C0051 BBa_B0030-BBa_C0078
  • Miniprep for all these parts.
  • Digestion for:
BBa_B0030 (S-P) BBa_E1010 (X-P) BBa_E0040 (X-P)
BBa_C0061 (X-P) BBa_C0051 (X-P) BBa_B0030-BBa_C0078 (X-S)
  • Gel run for BBa_B0030-BBa_C0078 to check for insert length: unfortunately, there was not a band where we expected...the only colony was a false positive. We'll try to ligate it in the next days.
  • Gel run for:
BBa_B0030 (S-P) BBa_E1010 (X-P) BBa_E0040 (X-P)
BBa_C0061 (X-P) BBa_C0051 (X-P)
  • Gel cut and DNA extraction.
  • We put DNA at -20°C. The next day we will perform some ligation reaction in different conditions, looking for the best protocol.



06/18/08

  • We planned the following ligation experiments:
    • Transformation with BBa_B0030(S-P), to check background noise (we will know the amount of not digested vector);
    • Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and no insert;
    • Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and no insert;
    • Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and BBa_C0061 insert;
    • Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0061 insert;
    • 3 Transformation with these ligations: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0051, BBa_E1010, BBa_E0040 inserts;
  • Antarctic Phosphatase for half of BBa_B0030 (S-P) volume.
  • We transformed 60 µl of TOP10 with 1 µl of BBa_B0030 (S-P)
  • We plated transformed bacteria and incubated them at 37°C overnight.
  • Antarctic Phosphatase for half a BBa_B0030 (S-P) volume.
  • Ligation (10 µl final volume):
    • BBa_B0030 alone
    • BBa_B0030 (no Ant.Phosph.) alone
    • BBa_B0030(no Ant.Phosph.)-BBa_C0061
    • BBa_B0030-BBa_C0061
    • BBa_B0030-BBa_C0078
    • BBa_B0030-BBa_E0040
    • BBa_B0030-BBa_E1010
  • We gave a lecture about Synthetic Biology and our current work at DIS (Department of Informatics and System Science).



06/19/08

  • We received sequencing results for:
    • BBa_J23100-BBa_E0240 (4 samples from 4 different colonies): all the 4 colonies were false positives
    • BBa_J23100-BBa_B0030: the sequence was correct!
    • BBa_R0051-BBa_B0030: the sequence was correct!
  • BBa_B0030(S-P) plate showed many colonies. We expect to find at least this amount of colonies in ligation plates.
  • We heated ligation at 65°C for 10 min to inactivate T4 Ligase.
  • We transformed 10 µl of the following ligations:
    • BBa_B0030 alone
    • BBa_B0030(no Ant.Phosph.) alone
    • BBa_B0030 (no Ant.Phosph.)-BBa_C0061
    • BBa_B0030-BBa_C0061
  • We didn't transform the other 3 ligations because we wanted to check plated transformation the next day, to save 3 agar plates if the experiment doesn't work.



06/20/08

  • Transformation results:
    • BBa_B0030 alone showed many colonies (less than BBa_B0030(S-P) seen the previous day)
    • BBa_B0030(no Ant.Phosph.) alone showed many colonies (the same quantity of BBa_B0030(S-P) seen the previous day)
    • BBa_B0030 (no Ant.Phosph.)-BBa_C0061 showed a carpet
    • BBa_B0030-BBa_C0061 showed a carpet
  • Now we are happy with these plates! Next week we will check insert length by colony PCR/electrophoresis.
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