Team:UNIPV-Pavia/Notebook/Week10

From 2008.igem.org

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*(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).
*(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).
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{|cellpadding="20px"
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|[[Image:pv_pcr_parallel1.jpg|thumb|300px|left|Thermal cycler working on BBa_J23100-BBa_B0030-BBa_C0012-'''BBa_B1006''' and BBa_B0030-BBa_I15009-BBa_BBa_B1006-'''BBa_R0082''' colonies: 2 min elongation]]
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|[[Image:pv_pcr_parallel2.jpg|thumb|300px|left|Thermal cycler working on **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 colonies: 3 min elongation]]
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*Electrophoresis (1.2% agarose) for BBa_J23100-BBa_B0030-BBa_C0012-'''BBa_B1006''' and BBa_B0030-BBa_I15009-BBa_BBa_B1006-'''BBa_R0082''':
*Electrophoresis (1.2% agarose) for BBa_J23100-BBa_B0030-BBa_C0012-'''BBa_B1006''' and BBa_B0030-BBa_I15009-BBa_BBa_B1006-'''BBa_R0082''':

Revision as of 14:21, 27 July 2008


Home.jpg Home Unipv logo.jpg The Team And.jpg The Project Safety.jpg Biological Safety Dna.png Parts Submitted to the Registry
Laptop.jpg Dry Lab Pipette.jpg Wet Lab Math.gif Modeling Note.jpg Protocols Notebook.gif Activity Notebook



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).
Thermal cycler working on BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 colonies: 2 min elongation
Thermal cycler working on **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 colonies: 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)
  • Gel results:
    • BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 1st colony
    • BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 1st colony
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 1st and 3rd colony
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 1st and 4th colony
  • We decided to keep two colonies 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 ligations because they are important parts for our project and we wanted to be sure they were correct.
  • We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.



07/25/08

  • Glycerol stocks/miniprep for:
    • BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1)
    • BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
  • We sent these purified plasmids:
    • BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
    • BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
  • to Primm for sequencing.
  • We put BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 plate at +4°C.
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