Team:UNIPV-Pavia/Notebook/Week10

From 2008.igem.org

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*Glycerol stocks/miniprep for:
*Glycerol stocks/miniprep for:
-
**J23100-BBa_B0030-BBa_C0012-'''BBa_B1006''' (1)
+
**J23100-B0030-C0012-'''B1006''' (1)
-
**BBa_B0030-BBa_I15009-BBa_BBa_B1006-'''BBa_R0082''' (1)
+
**B0030-I15009-B1006-'''R0082''' (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)
*We sent these purified plasmids:
*We sent these purified plasmids:
-
**BBa_B0030-BBa_I15009-BBa_BBa_B1006-'''BBa_R0082''' (1)
+
**B0030-I15009-B1006-'''R0082''' (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)
*to Primm for sequencing.
*to Primm for sequencing.
-
*We put BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' plate at +4°C.
+
*We put B0030-C0061-B0030-C0078-B1006-'''R0079''' plate at +4°C.

Revision as of 09:38, 2 October 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 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.
  • 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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