Team:UNIPV-Pavia/Notebook/Week15

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!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]
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'''08/25/08'''
'''08/25/08'''
<br>
<br>
-
*Pladmid digestion for:
+
*Plasmid digestion for:
-
**BBa_R0051 (S-P)
+
**R0051 (S-P)
-
**BBa_R0040 (S-P)
+
**R0040 (S-P)
-
**b (E-X)
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (E-X) (=Lig.b (E-X))
-
**BBa_B1006 (E-X)
+
**B1006 (E-X)
-
**12 (E-S)
+
**Lig.12 (E-S)
*Run/gel extraction.
*Run/gel extraction.
Line 66: Line 70:
**BBa_R0051 (S-P) - BBa_E0240 (X-P) (for promoter test)
**BBa_R0051 (S-P) - BBa_E0240 (X-P) (for promoter test)
**BBa_R0040 (S-P) - BBa_E0240 (X-P) (for promoter test)
**BBa_R0040 (S-P) - BBa_E0240 (X-P) (for promoter test)
-
**12 (E-S) - b (E-X) (for AND logic gate test)
+
**Lig.12 (E-S) - B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (E-X) (for AND logic gate test)
-
**12 (E-S) - BBa_B1006 (E-X) (to re-perform mutated assemblies)
+
**Lig.12 (E-S) - '''BBa_B1006 (E-X)''' (to re-perform mutated assemblies)
*We incubated ligations at 16°C overnight.
*We incubated ligations at 16°C overnight.
 +
 +
*We ordered 3OC6HSL (Sigma).
'''08/26/08'''
'''08/26/08'''
Line 76: Line 82:
'''08/27/08'''
'''08/27/08'''
<br>
<br>
-
*Plates grew correctly. We checked colony fluorescence under UV rays:
+
*Plates grew correctly. We checked colony fluorescence of three plates under UV rays:
-
**BBa_R0051-BBa_E0240 glowed
+
**'''R0051'''-E0240 glowed
-
**BBa_R0040-BBa_E0240 glowed
+
**'''R0040'''-E0240 glowed
-
**12-b (BBa_R0051-BBa_B0030-BBa_C0062-BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062-BBa_E0240) glowed
+
**Lig.12-Lig.b (R0051-B0030-C0062-B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006) glowed
 +
 
 +
*We picked up fluorescent colonies from these three plates to infect three 15 ml falcon tubes containing 1 ml of LB + Amp. We let the culture grow at 37°C, 220 rpm for 3 hours, then we prepared three 50 ul samples and watch them at microscope. Results are shown in The Project section (Experiments).
 +
 
 +
*Colony PCR for 5 colonies of Lig.12-'''B1006''' (called Lig.22).
 +
 
 +
{|
 +
|[[Image:pv_colonypcr_22again.jpg|thumb|370px|left|Lig.22 (5 colonies), Marker 1Kb]]
 +
|}
 +
 
 +
*Gel results: OK! we chose 1st colony to grow a 9 ml overnight culture.
 +
 
 +
'''08/28/08'''
 +
<br>
 +
*Glycerol stocks/miniprep for Lig.22.
 +
 
 +
*We sent purified plasmids to Primm for sequencing.
 +
 
 +
*We infected 9 ml of LB + Amp with 30 µl of R0062 glycerol stock.
 +
 
 +
'''08/29/08'''
 +
<br>
 +
*Glycerol stock/miniprep for R0062.

Latest revision as of 21:27, 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 15: 08/25/08 - 08/29/08

08/25/08

  • Plasmid digestion for:
    • R0051 (S-P)
    • R0040 (S-P)
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 (E-X) (=Lig.b (E-X))
    • B1006 (E-X)
    • Lig.12 (E-S)
  • Run/gel extraction.
  • Ligations:
    • BBa_R0051 (S-P) - BBa_E0240 (X-P) (for promoter test)
    • BBa_R0040 (S-P) - BBa_E0240 (X-P) (for promoter test)
    • Lig.12 (E-S) - B0030-C0061-B1006-R0062-B0030-E0040-B1006 (E-X) (for AND logic gate test)
    • Lig.12 (E-S) - BBa_B1006 (E-X) (to re-perform mutated assemblies)
  • We incubated ligations at 16°C overnight.
  • We ordered 3OC6HSL (Sigma).

08/26/08

  • We transformed/plated ligations.

08/27/08

  • Plates grew correctly. We checked colony fluorescence of three plates under UV rays:
    • R0051-E0240 glowed
    • R0040-E0240 glowed
    • Lig.12-Lig.b (R0051-B0030-C0062-B0030-C0061-B1006-R0062-B0030-E0040-B1006) glowed
  • We picked up fluorescent colonies from these three plates to infect three 15 ml falcon tubes containing 1 ml of LB + Amp. We let the culture grow at 37°C, 220 rpm for 3 hours, then we prepared three 50 ul samples and watch them at microscope. Results are shown in The Project section (Experiments).
  • Colony PCR for 5 colonies of Lig.12-B1006 (called Lig.22).
Lig.22 (5 colonies), Marker 1Kb
  • Gel results: OK! we chose 1st colony to grow a 9 ml overnight culture.

08/28/08

  • Glycerol stocks/miniprep for Lig.22.
  • We sent purified plasmids to Primm for sequencing.
  • We infected 9 ml of LB + Amp with 30 µl of R0062 glycerol stock.

08/29/08

  • Glycerol stock/miniprep for R0062.
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