Team:UNIPV-Pavia/Notebook/Week10

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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 10: 07/21/08 - 07/25/08

07/21/08

  • Colony PCR for BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079: 6 colonies from single colonies plate.
BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079
  • Gel results: all the 6 picked colonies were true positive! we chose the 5th colont to grow a 9 ml overnight culture.
  • We also infected 9 ml LB + Amp with 30 µl of BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0061 glycerol stocks to grow two overnight cultures.
  • Ligation:
    • BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1:2 ratio, 40 ng of vector)
    • BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (30 ng of vector)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
  • We incubated ligations at 16°C overnight.



07/22/08

  • Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.
  • Glycerol stocks/miniprep for:
BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 BBa_B0030-BBa_C0061
BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079
  • We sent BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 purified plasmids to Primm for sequencing.
  • Plasmid digestion for:
BBa_B0030-BBa_C0061 (E-S)
BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 (E-X)
  • Gel run/cut/gel extraction.



07/23/08

  • Ligation plates showed colonies!We put ligation plates at +4°C.
  • Ligation for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079.
  • We incubated ligation at 16°C overnight.



07/24/08

  • Transformation (1 µl) for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
  • Colony PCR for:
    • BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006
    • BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
  • (We performed one PCR for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
  • Electrophoresis (1.2% agarose) for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082:
Marker 1Kb, blank, BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (7 lanes), BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (6 lanes)
  • Electrophoresis (1% agarose) for BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 and BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006:
Marker 1Kb, BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (7 lanes), BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (7 lanes)