Team:UNIPV-Pavia/Notebook/Week7

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Notebook

Week 7: 06/30/08 - 06/5/08

06/30/08

• We received sequencing results for BBa_B0030-BBa_C0061 (4th colony and 7th colony): sequences were correct! We decided to keep the 4th colony.

• Colony PCR for BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0061: 10 colonies for every plate. Gel showed many working colonies: we chose first colonies for the two ligations.

Colony PCR for BBa_J23100-BBa_E0240, BBa_B0030-BBa_E0061: Marker and 10 colonies for each ligation

• We infected 9 ml LB + Amp with 30 µl of:

• Tomorrow we will be ready to perform 5 ligations!

07/1/08

• Glycerol stocks for:

• Miniprep for all these parts.

• We sent BBa_J23100-BBa_E0240 (1) and BBa_B0030-BBa_C0078 (1) to Primm for sequencing.

• We performed digestion for:

• Gel run/cut for:

• Gel extraction for these 5 parts.

• DNA precipitation with sodium acetate for:

• Ligation:
  • BBa_J23100-BBa_B0030-BBa_C0012
  • BBa_J23100-BBa_B0030-BBa_C0040
  • BBa_J23100-BBa_B0030-BBa_I15010
  • BBa_R0051-BBa_B0030-BBa_C0062
  • BBa_B0030-BBa_C0061-BBa_B1006

• We incubated ligation reaction at 16°C overnight.

07/2/08

• We transformed ligations (5 µl) and plated transformed bacteria.

• We also transformed BBa_B1006(E-X), BBa_J23100-BBa_B0030(S-P) and BBa_R0051-BBa_B0030 (1 µl) and plated transformed bacteria to estimate background noise.

• We received sequencing results for:
  • BBa_B0030-BBa_E0040
  • BBa_B0030-BBa_C0051
  • BBa_B0030-BBa_E1010
• All the sequences were correct!

• We infected 9 ml LB + Amp with 30 µl of:

• glycerol stocks.

07/3/08

• All the ligation plates showed carpets, while negative control plates showed a weak background noise.

• Single colonies plates for ligation plates.

• Glycerol stocks for:

• Miniprep for these parts.

• We performed digestion:

• Gel run/cut for:

• Gel extraction.

• DNA precipitation with sodium acetate for:

Ligation:

• • BBa_B0030-BBa_E0040-BBa_B1006
  • BBa_B0030-BBa_C0051-BBa_B0030
  • BBa_B0030-BBa_E1010-BBa_B1006

• We incubated ligation reactions at 16°C overnight.

07/4/08

• We transformed the 3 ligations (2 µl) and plated transformed bacteria.

• Colony PCR for single colonies plates (3 colonies for each plate):

• Electrophoresis for PCR result: unfortunately, BBa_J23100-BBa_B0030-BBa_C0012 and BBa_J23100-BBa_B0030-BBa_I15010 did not show any true positive colony. We decided to re-perform colony PCR for these two parts next week. We chose to keep the first colonies for the other 3 ligations to grow 9 ml cultures overnight.

Colony PCR: Marker (1), BBa_J23100-BBa_B0030-BBa_C0012 (2-4), BBa_J23100-BBa_B0030-BBa_C0040 (5-7), BBa_J23100-BBa_B0030-BBa_I15010 (8-10), BBa_R0051-BBa_B0030-BBa_C0062 (11-13), BBa_B0030-BBa_C0061-BBa_B1006 (14-16), blank (17)

07/5/08

• All the ligation plates showed colonies! Next week we will perform colony PCR to find true positive colonies.

• Glycerol stocks and Miniprep for: BBa_J23100-BBa_B0030-BBa_C0040 (1), BBa_R0051-BBa_B0030-BBa_C0062 (1), BBa_B0030-BBa_C0061-BBa_B1006 (1).

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