Team:UNIPV-Pavia/Notebook/Week10

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*We decided to keep two colonies for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
*We decided to keep two colonies for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
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*The fifth colony of B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.
*We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.
*We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.

Revision as of 17:50, 26 October 2008


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

07/21/08

  • Colony PCR for B0030-C0078-B1006-R0079: 6 colonies from single colonies plate.
B0030-C0078-B1006-R0079
  • Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.
  • We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-R0040 and B0030-C0061 glycerol stocks to grow two overnight cultures.
  • Ligation:
    • J23100-B0030-C0012-B1006 (1:2 ratio, 40 ng of vector)
    • B0030-I15009-B1006-R0082 (30 ng of vector)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-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:
J23100-B0030-C0040-B1006-R0040 B0030-C0061
B0030-C0078-B1006-R0079
  • We sent J23100-B0030-C0040-B1006-R0040 and B0030-C0078-B1006-R0079 purified plasmids to Primm for sequencing.
  • Plasmid digestion for:
B0030-C0061 (E-S) B0030-C0078-B1006-R0079 (E-X)
  • Gel run/cut/gel extraction.



07/23/08

  • Ligation plates showed colonies!We put ligation plates at +4°C.
  • Ligation for B0030-C0061-B0030-C0078-B1006-R0079.
  • We incubated ligation at 16°C overnight.



07/24/08

  • Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
  • Colony PCR for:
    • J23100-B0030-C0012-B1006
    • B0030-I15009-B1006-R0082
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
  • (We performed one PCR for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
Thermal cycler working on J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 colonies: 2 min elongation
Thermal cycler working on B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 colonies: 3 min elongation
  • Electrophoresis (1.2% agarose) for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082:
Marker 1Kb, blank, J23100-B0030-C0012-B1006 (7 lanes), B0030-I15009-B1006-R0082 (6 lanes)
  • Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006:
Marker 1Kb, B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (7 lanes), B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (7 lanes)
  • Gel results:
    • J23100-B0030-C0012-B1006 1st colony
    • B0030-I15009-B1006-R0082 1st colony
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 1st and 4th colony
  • We decided to keep two colonies for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
  • The fifth colony of B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.
  • We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.



07/25/08

  • Glycerol stocks/miniprep for:
    • J23100-B0030-C0012-B1006 (1)
    • B0030-I15009-B1006-R0082 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
  • We sent these purified plasmids:
    • B0030-I15009-B1006-R0082 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
  • to Primm for sequencing.
  • We put B0030-C0061-B0030-C0078-B1006-R0079 plate at +4°C.
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