Team:UNIPV-Pavia/Notebook/Week5

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Latest revision as of 21:25, 26 October 2008

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Notebook

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:

• glycerol stocks. We incubated the 9 ml culture overnight at 37°C, 220 rpm.

06/17/08

• Glycerol stocks for:

• Miniprep for all these parts.

• Digestion for:

• 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:

• 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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