Team:Paris-Saclay/Project/Notebook/Week 11

From 2012.igem.org

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[[Category:Team:Paris-Saclay/Project Gemote/Notebook|5]]
[[Category:Team:Paris-Saclay/Project Gemote/Notebook|5]]
__NOTOC__
__NOTOC__
-
====6th August====
+
====13th August====
-
 
+
-
*Sending of the B1 sample to sequencing with two pairs of primers
+
-
**K-274100 Forward and Reverse
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-
**Plasmid pSB1A2 Forward and Reverse
+
-
 
+
-
 
+
-
====7th August====
+
-
 
+
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*Liquid culture of B1 in order to prepare a glycerol stock
+
-
**Receipt of new primers
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-
**2 Forward
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-
**3 Reverse
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-
**Plasmid Reverse
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-
 
+
-
 
+
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====8th August====
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{| width="85%"
{| width="85%"
|-
|-
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| style="width: 50%;"| Amplification of the plasmid pSB1A2, BBa_K274100 and BBa_K115017 by PCR with the new primers. Visualization by electrophoresis on a 2% Agarose gel for BBa_K115017 and a 0.8% Agarose gel for BBa_K274100 and the plasmid pSB1A2. We are expecting a band at 147 bp for BBa_K115017, 3408 bp for BBa_K274100 and 2079 for the plasmid.
+
| style="width: 50%;"|Purification by PCR clean-up of BBa_K115017 treated with DPNI. To visualize the fragment, an electrophoresis has been made with a 2% Agarose gel. We are expecting a band at the size of 123 bp.
-
| style="width: 35%;"| [[File:Week10-2.jpg|right|330px]]
+
| style="width: 35%;"| [[File:Week11-1.jpg|right|330px]]
 +
|-
 +
| style="width: 50%;"|[[File:Week11-2.jpg|left|330px]]
 +
| style="width: 35%;"| New PCR of BBa_K115017 to obtain more quantity of the fragment
|}
|}
PCR program used:
PCR program used:
-
[[File:Week10-3.jpg|600px]]
+
[[File:Week11-3.jpg|600px]]
-
====9th August====
+
*Digestion by DPNI of the BBa_K115017 in order to degrade the matrix plasmid.
{| width="85%"
{| width="85%"
|-
|-
-
| style="width: 50%;"|New amplification by PCR of the plasmid pSB1A2, BBa_K274100 and BBa_K115017 as it has been done the day before.
+
| style="width: 50%;"|Determining the concentration of the plasmid pSB1A2 which has been linearized by HINDIII. We are expecting a band at the size of (2079+935) ~3kbp
-
| style="width: 35%;"| [[File:Week10-4.jpg|right|330px]]
+
| style="width: 35%;"| [[File:Week11-4.jpg|right|330px]]
 +
|-
 +
| style="width: 50%;"|A new PCR of the plasmid pSB1A2 is realized to obtain more quantity of the plasmid. We are expecting a band at the size of 2079 bp.
 +
| style="width: 35%;"| [[File:Week11-5.jpg|right|330px]]
|}
|}
-
*Digestion by EcoRI of the plasmid that contains BBa_K274100.
+
PCR program used:
 +
[[File:Week11-6.jpg|600px]]
-
====10th August====
+
====14th August====
{| width="85%"
{| width="85%"
|-
|-
-
| style="width: 50%;"|Amplification by PCR of BBa_K274100 already digested by EcoRI. Visualization by electrophoresis on a 0.8% Agarose gel.
+
| style="width: 50%;"|Determining of the concentration of the BBa_K274100 by electrophoresis on a gel at O.8% Agarose. We are expecting a band at the size of 2100bp
-
| style="width: 35%;"| [[File:Week10-5.jpg|right|300px]]
+
| style="width: 35%;"| [[File:Week11-7.jpg|right|330px]]
|}
|}
 +
*Digestion of the BBa_K274100 by DPNI to degrade the matrix plasmid.
 +
*Stocking up cells with glycerol
 +
**PSB1A3Amil CP + Ampicilline
 +
**PSB1C3 Amil GFP + Chloramphinicol
-
PCR program used:
+
 
-
[[File:Week10-8.jpg|400px]]
+
 
 +
====15th August====
 +
 
 +
Day of public holiday in France.
 +
 
 +
 
 +
====16th August====
{| width="85%"
{| width="85%"
|-
|-
-
| style="width: 50%;"|Miniprep of BBa_K274100 followed by Nanovue to determine the concentration of the sample
+
| style="width: 50%;"|PCR of BBa_K274100 and visualization by 0.8% Agarose gel electrophoresis. We are expecting a band at 3400bp.
-
| style="width: 35%;"| [[File:Week10-6.jpg|right|330px]]
+
| style="width: 35%;"| [[File:Week11-8.jpg|right|330px]]
-
|-
+
-
| style="width: 50%;"|Miniprep of the Plasmid pSB1A2 followed by Nanovue to determine the concentration of the sample
+
-
| style="width: 35%;"| [[File:Week10-7.jpg|right|330px]]
+
|}
|}
-
*Digestion by HindIII of the plasmid pSB1A2 in order to linearize it.
+
PCR program used:
 +
[[File:Week11-9.jpg|600px]]
 +
 
 +
*Digestion of the BBa_K274100 by DPNI to degrade the matrix plasmid.
 +
 
 +
====17th august====
{| width="85%"
{| width="85%"
|-
|-
-
| style="width: 50%;"|Digestion of BBa_K115017 by DPNI to eliminate the plasmid matrix. Visualization by electrophoresis on a 2% Agarose gel. We are expecting a band at 123 bp.
+
| style="width: 50%;"|Visualization of the DPNI’s digestion of the plasmid pSB1A2 by 0.8% Agarose gel electrophoresis. . We are expecting a band at the size of 2079 bp.
-
| style="width: 35%;"| [[File:Week10-1.jpg|right|280px]]
+
| style="width: 35%;"| [[File:Week11-10.jpg|right|330px]]
|}
|}
 +
 +
*Miniprep of the plasmid pSB1A2.
 +
*Miniprep of BBa_K274100 and BBa_K115017
 +
*Gibson assembly of the B construction
 +
*Transformation of DH5α competent cells with the Gibson B construction. The Petri dishes are placed at 37°C.
{{Team:Paris-Saclay/Follow}}
{{Team:Paris-Saclay/Follow}}

Revision as of 06:25, 26 September 2012

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13th August

Purification by PCR clean-up of BBa_K115017 treated with DPNI. To visualize the fragment, an electrophoresis has been made with a 2% Agarose gel. We are expecting a band at the size of 123 bp.
Week11-1.jpg
Week11-2.jpg
 New PCR of BBa_K115017 to obtain more quantity of the fragment


PCR program used: Week11-3.jpg

  • Digestion by DPNI of the BBa_K115017 in order to degrade the matrix plasmid.
Determining the concentration of the plasmid pSB1A2 which has been linearized by HINDIII. We are expecting a band at the size of (2079+935) ~3kbp
Week11-4.jpg
A new PCR of the plasmid pSB1A2 is realized to obtain more quantity of the plasmid. We are expecting a band at the size of 2079 bp.
Week11-5.jpg

PCR program used: Week11-6.jpg


14th August

Determining of the concentration of the BBa_K274100 by electrophoresis on a gel at O.8% Agarose. We are expecting a band at the size of 2100bp
Week11-7.jpg
  • Digestion of the BBa_K274100 by DPNI to degrade the matrix plasmid.
  • Stocking up cells with glycerol
    • PSB1A3Amil CP + Ampicilline
    • PSB1C3 Amil GFP + Chloramphinicol


15th August

Day of public holiday in France.


16th August

PCR of BBa_K274100 and visualization by 0.8% Agarose gel electrophoresis. We are expecting a band at 3400bp.
Week11-8.jpg

PCR program used: Week11-9.jpg

  • Digestion of the BBa_K274100 by DPNI to degrade the matrix plasmid.

17th august

Visualization of the DPNI’s digestion of the plasmid pSB1A2 by 0.8% Agarose gel electrophoresis. . We are expecting a band at the size of 2079 bp.
Week11-10.jpg
  • Miniprep of the plasmid pSB1A2.
  • Miniprep of BBa_K274100 and BBa_K115017
  • Gibson assembly of the B construction
  • Transformation of DH5α competent cells with the Gibson B construction. The Petri dishes are placed at 37°C.

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