Team:Paris Saclay/Notebook/August/22

From 2014.igem.org

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=Friday 22nd August=
=Friday 22nd August=
==Lab work==
==Lab work==
-
===D - Lemon Scent===
+
===Lemon Scent===
====pPSI digestion====
====pPSI digestion====
-
40µl pPSI
 
-
2µl H2O
+
{| class="wikitable centre" width="25%"
-
 
+
|+ 
-
2µL PacI
+
|-
-
 
+
! scope=col | components
-
5µL FAST digest buffer
+
! scope=col | volumes
 +
|-
 +
|pPSI
 +
|40μl
 +
|-
 +
|FastDigest green buffer 10X
 +
|5μl
 +
|-
 +
|PacI
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|2µl
 +
|}
====pPSI dephosphorylation====
====pPSI dephosphorylation====
 +
 +
[[File:Paris_Saclay_140822.jpg|left]]
After 2 hours, add 1.5µL of Alkaline Phosphatase (AP) and let it 1 hour.
After 2 hours, add 1.5µL of Alkaline Phosphatase (AP) and let it 1 hour.
Line 18: Line 32:
Then, inactivation of AP at 65°C during 15 minutes.
Then, inactivation of AP at 65°C during 15 minutes.
-
We've made a electrophoresis to chech the digestion.
+
We've made a electrophoresis to check the digestion.
-
(photo)
+
 
 +
'''Legend'''
 +
#pPSI+Apra digested via PacI, dephosphorylated 5µl
 +
#pPSI+Apra digested via PacI, dephosphorylated 5µl
 +
#ladder 5µl
 +
 
 +
==== Ligation of BBa_K517003, BBa_K762100, and Geraniol synthase in pPSI====
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ BBa_K517003
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|BBa_K517003
 +
|10μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|-
 +
|ligase
 +
|1µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ BBa_K5762100
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|BBa_K762100
 +
|10μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|-
 +
|ligase
 +
|1µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ Geraniol Synthase (GS)
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|GS
 +
|10μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|-
 +
|ligase
 +
|1µl
 +
|}
 +
 
 +
And let it in the freezer for 3 hours.
 +
 
 +
====Transformation in competent E.coli DH5α====
 +
 
 +
After 3 hours, take out the ligation tubes and add 100 µl of competent DH5α in each tube.
 +
 
 +
Then proceed to the transformation [https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_competent_e.coli_by_CaCl2 protocol].
 +
 
 +
- 20 minutes at 4°C
 +
 
 +
- 2 minutes 30 seconds at 42°C
 +
 
 +
- 2 minutes at 4 °C
 +
 
 +
- Add 0.9 ml of LB into the tubes
 +
 
 +
Finally put it in a incubator at 37°C for 30 minutes
 +
 
 +
 
 +
 
 +
====PCR of CAD ====
 +
With or synthetic gene, we decided to do a PCR to have more matrice.
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ In one tube
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|H<sub>2</sub>O
 +
|27μl
 +
|-
 +
|5x phusion buffer
 +
|2µl
 +
|-
 +
|dNTP 10 mM
 +
|2µl
 +
|-
 +
|iPS83 10µM
 +
|5µl
 +
|-
 +
|iPS84 10µM
 +
|5µl
 +
|-
 +
|DMSO
 +
|1.5µl
 +
|}
 +
 
 +
====PCR purification====
 +
We use the kit PCR clean-up
 +
 
 +
the elution volume is 20µl
-
==== Ligation of LS/GS/PS in pPSI====
+
{| class="wikitable centre" width="25%"
-
For each synthase,put:
+
|+ kit PCR clean-up
-
#10 µL of synthase
+
! scope=col | DNA Polymerase
-
#2 µL of pPSI
+
! scope=col | volume
-
#2 µL of buffer
+
|-
-
#5 µL of water
+
|Phusion
-
#1 µL of ligase
+
|0.5µl
 +
|-
 +
|}
-
And let it in the freezer during 3 hours.
+
{| class="wikitable centre" width="25%"
 +
|+ PCR cycle
 +
|-
 +
! scope=col | Temperature (°C)
 +
! scope=col | time
 +
|-
 +
|98
 +
|30 sec
 +
|-
 +
|98
 +
|10 sec
 +
|-
 +
|58
 +
|30 sec
 +
|-
 +
|72
 +
|45 sec
 +
|-
 +
|72
 +
|10 min
 +
|-
 +
|}
 +
==Photo of the Day==
 +
[[File:Paris Saclay 22_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 15:10, 14 October 2014

Contents

Friday 22nd August

Lab work

Lemon Scent

pPSI digestion

components volumes
pPSI 40μl
FastDigest green buffer 10X 5μl
PacI 2µl
H2O 2µl

pPSI dephosphorylation

Paris Saclay 140822.jpg

After 2 hours, add 1.5µL of Alkaline Phosphatase (AP) and let it 1 hour.

Then, inactivation of AP at 65°C during 15 minutes.

We've made a electrophoresis to check the digestion.

Legend

  1. pPSI+Apra digested via PacI, dephosphorylated 5µl
  2. pPSI+Apra digested via PacI, dephosphorylated 5µl
  3. ladder 5µl

Ligation of BBa_K517003, BBa_K762100, and Geraniol synthase in pPSI

BBa_K517003
components volumes
BBa_K517003 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl
BBa_K5762100
components volumes
BBa_K762100 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl
Geraniol Synthase (GS)
components volumes
GS 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl

And let it in the freezer for 3 hours.

Transformation in competent E.coli DH5α

After 3 hours, take out the ligation tubes and add 100 µl of competent DH5α in each tube.

Then proceed to the transformation protocol.

- 20 minutes at 4°C

- 2 minutes 30 seconds at 42°C

- 2 minutes at 4 °C

- Add 0.9 ml of LB into the tubes

Finally put it in a incubator at 37°C for 30 minutes


PCR of CAD

With or synthetic gene, we decided to do a PCR to have more matrice.

In one tube
components volumes
H2O 27μl
5x phusion buffer 2µl
dNTP 10 mM 2µl
iPS83 10µM 5µl
iPS84 10µM 5µl
DMSO 1.5µl

PCR purification

We use the kit PCR clean-up

the elution volume is 20µl

kit PCR clean-up
DNA Polymerase volume
Phusion 0.5µl
PCR cycle
Temperature (°C) time
98 30 sec
98 10 sec
58 30 sec
72 45 sec
72 10 min

Photo of the Day

600px

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/22"