Team:Paris Saclay/Notebook/August/27

From 2014.igem.org

(Difference between revisions)
(→Ligation)
(→B - Construction of the fusion protein (color))
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==Lab Work==
==Lab Work==
===B - Construction of the fusion protein (color)===
===B - Construction of the fusion protein (color)===
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====PCR clean-up of colonies containing pGEMTeasy+chromoprotein====
+
 
 +
==== Electrophoresis of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI ====
 +
''by Laetitia''
 +
 
 +
Digestion made the 26th by Laetitia. After the migration on agarose gel (100V), we cut the band corresponding to the chromoprotein gene on UV table.
 +
 
 +
 
 +
====Purification of the chromoprotein gene from the agarose gel ====
''by Sean''
''by Sean''
-
Three colonies were selected from the gel prepared earlier by Laetitia, namely #3,#4, and #6. The three gel samples underwent PCR clean-up.
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We used the kit PCR-Clean-UP to extract the DNA from the agarose gel. Elution volume : 15µl
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====Gel electrophoresis of PCR clean-up results containing pGEMTeasy+chromoprotein====
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 +
====Gel electrophoresis of the purification product ====
''by Sean''
''by Sean''
 +
 +
The goal is to check if the purification of the chromoprotein gene have worked.
PCR clean-up was performed ealier [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/27#PCR_clean-up_of_colonies_containing_pGEMTeasy.2Bchromoprotein today]. 1µl of each result was used for the electrophoresis. Nothing appeared under UV.
PCR clean-up was performed ealier [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/27#PCR_clean-up_of_colonies_containing_pGEMTeasy.2Bchromoprotein today]. 1µl of each result was used for the electrophoresis. Nothing appeared under UV.
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*pCFP+
*pCFP+
*pYFP+
*pYFP+
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===D - lemon scent ===
===D - lemon scent ===

Revision as of 15:03, 28 August 2014

Contents

Wednesday 27th August

Lab Work

B - Construction of the fusion protein (color)

Electrophoresis of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI

by Laetitia

Digestion made the 26th by Laetitia. After the migration on agarose gel (100V), we cut the band corresponding to the chromoprotein gene on UV table.


Purification of the chromoprotein gene from the agarose gel

by Sean

We used the kit PCR-Clean-UP to extract the DNA from the agarose gel. Elution volume : 15µl


Gel electrophoresis of the purification product

by Sean

The goal is to check if the purification of the chromoprotein gene have worked.

PCR clean-up was performed ealier today. 1µl of each result was used for the electrophoresis. Nothing appeared under UV.

Ligation

by Sean

Transformation of odor free E. coli with plasmids coding Fluo Protein

by Terry

Our fusion protein was not expressed in pGEMTeasy (no color in the culture), so, if our system do not work at all, I'm preparing FP (Fluorescent Protein) for our lemon.

6 plamids coding FP have been transformed in Odor Free :

  • pEYFP
  • pGFP
  • pESFP
  • pRFP+
  • pCFP+
  • pYFP+

D - lemon scent

Plasmide extraction

By Mélanie

Extraction of pPS5 with the phenol protocol (as previously described)

PCR of Limonene synthase (BBa762100)

by Mélanie

5 tubes to do a gradient

components volumes
H2O 29.75μl
Gotaq buffer 10µl
dNTP 10mM 1µl
iPS67 1µM
iPS66 1µM
DNA 1µl
MgCl2 4µl
Gotaq 1.25µl
PCR cycle
step temperature (°C) time
1 95 2min
2 95 30 sec
3 49-55 (gradient) 30sec
4 72 2min
5 72 5min

Electrophoresis of pPS3 and pPS4

by Terry

Result from yesterday 's extraction.

Alice, donne moi la photo.

Back to the calendar

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