Team:UNIPV-Pavia/Notebook/Week7

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!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]
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|-
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!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]
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!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]
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!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]
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!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]
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!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]
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!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]
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!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]
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|-
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!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]
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!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]
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!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]
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*We received sequencing results for '''BBa_B0030'''-BBa_C0061 (4th colony and 7th colony): sequences were correct! We decided to keep the 4th colony.
*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 and '''BBa_B0030'''-BBa_C0061: 5 colonies for every plate. Gel showed many working colonies: we chose first colonies for the two ligations.
{|
{|
-
|[[Image:pv_pcr_01_08.jpg|thumb|300px|left|Colony PCR for '''BBa_J23100'''-BBa_E0240, '''BBa_B0030'''-BBa_E0061: Marker and 10 colonies for each ligation]]
+
|[[Image:pv_pcr_01_08.jpg|thumb|470px|left|Colony PCR for '''BBa_J23100'''-BBa_E0240, '''BBa_B0030'''-BBa_E0061: Marker and 5 colonies for each ligation]]
|}
|}
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<br><br>
<br><br>
-
'''07/1/08'''
+
'''07/2/08'''
<br>
<br>
 +
*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:
 +
{|cellpadding="20px"
 +
|BBa_B0030
 +
|'''BBa_B0030'''-BBa_E0040
 +
|'''BBa_B0030'''-BBa_C0051
 +
|-
 +
|BBa_B1006
 +
|'''BBa_B0030'''-BBa_E1010
 +
|}
 +
*glycerol stocks.
 +
 +
<br><br>
 +
'''07/3/08'''
 +
<br>
 +
*All the ligation plates showed carpets, while negative control plates showed a weak background noise.
 +
 +
*Single colonies plates for ligation plates.
 +
 +
*Glycerol stocks for:
 +
{|cellpadding="20px"
 +
|BBa_B0030
 +
|'''BBa_B0030'''-BBa_E0040
 +
|'''BBa_B0030'''-BBa_C0051
 +
|-
 +
|BBa_B1006
 +
|'''BBa_B0030'''-BBa_E1010
 +
|}
 +
 +
*Miniprep for these parts.
 +
 +
*We performed digestion:
 +
{|cellpadding="20px"
 +
|BBa_B0030 (E-X)
 +
|'''BBa_B0030'''-BBa_E0040 (E-S)
 +
|'''BBa_B0030'''-BBa_C0051 (E-S)
 +
|-
 +
|BBa_B1006 (E-X)
 +
|'''BBa_B0030'''-BBa_E1010 (E-S)
 +
|}
 +
 +
*Gel run/cut for:
 +
{|cellpadding="20px"
 +
|'''BBa_B0030'''-BBa_E0040 (E-S)
 +
|'''BBa_B0030'''-BBa_C0051 (E-S)
 +
|'''BBa_B0030'''-BBa_E1010 (E-S)
 +
|}
 +
 +
*Gel extraction.
 +
 +
*DNA precipitation with sodium acetate for:
 +
{|cellpadding="20px"
 +
|BBa_B0030 (E-X)
 +
|BBa_B1006 (E-X)
 +
|}
 +
 +
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.
 +
 +
<br><br>
 +
'''07/4/08'''
 +
<br>
 +
*We transformed the 3 ligations (2 µl) and plated transformed bacteria.
 +
 +
*Colony PCR for single colonies plates (3 colonies for each plate):
 +
{|cellpadding="20px"
 +
|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
 +
|}
 +
 +
*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.
 +
 +
{|
 +
|[[Image:pv_pcr_09_10_11_12_16.jpg|thumb|450px|left|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)]]
 +
|}
 +
 +
<br><br>
 +
'''07/5/08'''
 +
<br>
 +
*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).

Latest revision as of 21:26, 26 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 22 Week 23 Week 24



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: 5 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 5 colonies for each ligation
  • We infected 9 ml LB + Amp with 30 µl of:
BBa_C0012 BBa_B1006 BBa_R0051-BBa_B0030 BBa_B0030-BBa_C0061
BBa_C0062 BBa_J23100-BBa_E0240 (1) BBa_B0030-BBa_C0078 (1) BBa_J23100-BBa_B0030
BBa_C0040 BBa_I15010
  • Tomorrow we will be ready to perform 5 ligations!



07/1/08

  • Glycerol stocks for:
BBa_C0012 BBa_B1006 BBa_R0051-BBa_B0030 BBa_B0030-BBa_C0061
BBa_C0062 BBa_J23100-BBa_E0240 (1) BBa_B0030-BBa_C0078 (1) BBa_J23100-BBa_B0030
BBa_C0040 BBa_I15010
  • 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:
BBa_C0012 (X-P) BBa_B1006 (E-X) BBa_R0051-BBa_B0030 (S-P) BBa_B0030-BBa_C0061 (E-S)
BBa_C0062 (X-P) BBa_C0040 (X-P) BBa_I15010 (X-P) BBa_J23100-BBa_B0030 (S-P)
  • Gel run/cut for:
BBa_C0012 (X-P) BBa_C0062 (X-P) BBa_B0030-BBa_C0061 (E-S)
BBa_C0040 (X-P) BBa_I15010 (X-P)
  • Gel extraction for these 5 parts.
  • DNA precipitation with sodium acetate for:
BBa_B1006 (E-X) BBa_R0051-BBa_B0030 (S-P) BBa_J23100-BBa_B0030 (S-P)
  • 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:
BBa_B0030 BBa_B0030-BBa_E0040 BBa_B0030-BBa_C0051
BBa_B1006 BBa_B0030-BBa_E1010
  • 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:
BBa_B0030 BBa_B0030-BBa_E0040 BBa_B0030-BBa_C0051
BBa_B1006 BBa_B0030-BBa_E1010
  • Miniprep for these parts.
  • We performed digestion:
BBa_B0030 (E-X) BBa_B0030-BBa_E0040 (E-S) BBa_B0030-BBa_C0051 (E-S)
BBa_B1006 (E-X) BBa_B0030-BBa_E1010 (E-S)
  • Gel run/cut for:
BBa_B0030-BBa_E0040 (E-S) BBa_B0030-BBa_C0051 (E-S) BBa_B0030-BBa_E1010 (E-S)
  • Gel extraction.
  • DNA precipitation with sodium acetate for:
BBa_B0030 (E-X) BBa_B1006 (E-X)

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):
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
  • 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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