Team:Paris Saclay/Notebook/August/19

From 2014.igem.org

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(→Purification of PCR products of limonen synthase)
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==Lab work==
==Lab work==
-
===D - Lemon Scent===
+
===Chromoprotein + Lemon scent===
 +
Electrophoresis with the PpsI plasmide (obtain on thursday) + the chromoprotein PCR:
 +
we obtain results as expected
 +
 
 +
#ladder 10µl
 +
#pPSI 1.2µl
 +
#pPSI 1.2µl
 +
#pPSI 1.2µl
 +
#pPSI 1.2µl
 +
#pPSI 1.2µl
 +
#p cola 1.2µl
 +
#PCR chromoprotein 2µl
 +
 
 +
[[File:Paris_Saclay_140819.jpg]]
 +
 
 +
===Lemon Scent===
==== Digestion of PCR results  ====
==== Digestion of PCR results  ====
Line 64: Line 79:
|2 µl
|2 µl
|}
|}
-
 
{| class="wikitable centre" width="50%"
{| class="wikitable centre" width="50%"
|+   
|+   
-
|-
+
|-''Italic text''
! scope=col | components
! scope=col | components
! scope=col | volumes
! scope=col | volumes
|-
|-
-
|PCR purified pPS1
+
|Purified pPS1
|30 μl
|30 μl
|-
|-
Line 84: Line 98:
|4 µl
|4 µl
|}
|}
-
 
====Purification of PCR products of limonen synthase====
====Purification of PCR products of limonen synthase====
 +
''by Laetitia''
 +
We used the PCR products of limonen synthase prepared previously : [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/18 August 18th]
We used the PCR products of limonen synthase prepared previously : [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/18 August 18th]
The five samples were pooled in one and then purified using this protocol : [https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up protocol]
The five samples were pooled in one and then purified using this protocol : [https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up protocol]
 +
====Bacterial transformation====
 +
''by Laetitia''
 +
 +
We used DH5alpha competent bacteria (prepared the 14/08) and they were transformed using the ligation product prepared previously  [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/18 August 18th] using this protocol : [https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_supercompetent_Ecoli_cells_with_CaCl2 protocol]
 +
 +
We transformed 100µL of competent bacteria and we added the totality of the ligation product (pGEMTeasy containing LS or PS or GS)
 +
 +
====Preparation of petri dishes====
 +
''by Terry''
 +
 +
{| class="wikitable centre" width="25%"
 +
|+ 10 dishes with:
 +
|-
 +
! scope=col | Antibiotic
 +
! scope=col | Concentration
 +
|-
 +
|XGal
 +
|80μg/ml
 +
|-
 +
|IPTG
 +
|2*10<sup>-3</sup>
 +
|-
 +
|Ampicillin
 +
|10<sup>-3</sup>
 +
|}
 +
 +
{| class="wikitable centre" width="25%"
 +
|+ 4 dishes with:
 +
|-
 +
! scope=col | Antibiotic
 +
! scope=col | Concentration
 +
|-
 +
|Ampicillin
 +
|4*10<sup>-4</sup>
 +
|}
 +
 +
====Bacterial culture====
 +
''by Laetitia''
 +
 +
After the bacterial transformation, we spread for each strain (LS or PS or GS):
 +
 +
{| class="wikitable centre" width="25%" style="text-align:center"
 +
|+
 +
|-
 +
! scope=col | dish type
 +
! scope=col | 100µl
 +
! scope=col | 200µl
 +
! scope=col | concentrate
 +
! scope=col | control
 +
|-
 +
|Ampicillin
 +
|X
 +
|
 +
|
 +
|
 +
|-
 +
|Ampicillin+IPTG+XGal
 +
|X
 +
|X
 +
|X
 +
|X
 +
|}
 +
 +
We leave the dishes overnight at 37°C.
 +
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 19_august.jpg|600px|center]]
 +
 +
'''Members present:'''
 +
* Instructors and advisors: Alice, Solenne and Sylvie.
 +
* Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:02, 14 October 2014

Contents

Tuesday 19th August

Lab work

Chromoprotein + Lemon scent

Electrophoresis with the PpsI plasmide (obtain on thursday) + the chromoprotein PCR: we obtain results as expected

  1. ladder 10µl
  2. pPSI 1.2µl
  3. pPSI 1.2µl
  4. pPSI 1.2µl
  5. pPSI 1.2µl
  6. pPSI 1.2µl
  7. p cola 1.2µl
  8. PCR chromoprotein 2µl

Paris Saclay 140819.jpg

Lemon Scent

Digestion of PCR results

by Sean, Hoang Vu, Eugene

components volumes
PCR purified LS 15 μl
FastDigest green buffer 10X 2 μl
PacI 1 µl
H2O 2 µl
components volumes
PCR purified GS 15 μl
FastDigest green buffer 10X 2 μl
PacI 1 µl
H2O 2 µl
components volumes
PCR purified PS 15 μl
FastDigest green buffer 10X 2 μl
PacI 1 µl
H2O 2 µl
components volumes
Purified pPS1 30 μl
FastDigest green buffer 10X 4 μl
PacI 2 µl
H2O 4 µl

Purification of PCR products of limonen synthase

by Laetitia

We used the PCR products of limonen synthase prepared previously : August 18th

The five samples were pooled in one and then purified using this protocol : protocol

Bacterial transformation

by Laetitia

We used DH5alpha competent bacteria (prepared the 14/08) and they were transformed using the ligation product prepared previously August 18th using this protocol : protocol

We transformed 100µL of competent bacteria and we added the totality of the ligation product (pGEMTeasy containing LS or PS or GS)

Preparation of petri dishes

by Terry

10 dishes with:
Antibiotic Concentration
XGal 80μg/ml
IPTG 2*10-3
Ampicillin 10-3
4 dishes with:
Antibiotic Concentration
Ampicillin 4*10-4

Bacterial culture

by Laetitia

After the bacterial transformation, we spread for each strain (LS or PS or GS):

dish type 100µl 200µl concentrate control
Ampicillin X
Ampicillin+IPTG+XGal X X X X

We leave the dishes overnight at 37°C.


Photo of the Day

Paris Saclay 19 august.jpg

Members present:

  • Instructors and advisors: Alice, Solenne and Sylvie.
  • Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/19"