Team:Paris Saclay/Notebook/August/20

From 2014.igem.org

(Difference between revisions)
(→Digestion of CAD and PMCS5)
m (→Photo of the Day)
 
(27 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Paris_Saclay/notebook_header}}
{{Team:Paris_Saclay/notebook_header}}
=Wednesday 20th August=
=Wednesday 20th August=
-
{{Team:Paris_Saclay/notebook_footer}}
+
==Lab work==
 +
====Petri dishes====
 +
''by Laetitia''
 +
 
 +
{| 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>
 +
|}
 +
 
 +
===Construction of the fusion protein (color)===
 +
====PCR====
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+ PCR of chromoprotein
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|DNA
 +
|.5µl
 +
|-
 +
|GoTaq buffer 5X
 +
|10μl
 +
|-
 +
|DMSO
 +
|1.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|22.75µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|4µl
 +
|-
 +
|dNTP
 +
|1µl
 +
|-
 +
|iPS83
 +
|5µl
 +
|-
 +
|iPS84
 +
|5µl
 +
|-
 +
|GoTaq polymerase
 +
|.25µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+ PCR cycle
 +
|-
 +
! scope=col | step
 +
! scope=col | temperature (°C)
 +
! scope=col | time (min)
 +
|-
 +
|1
 +
|95
 +
|2
 +
|-
 +
|2
 +
|95
 +
|.5
 +
|-
 +
|3
 +
|58
 +
|.5
 +
|-
 +
|4
 +
|72
 +
|1.75
 +
|-
 +
|5
 +
|72
 +
|5
 +
|}
 +
 
 +
====Cloning in pGEMTesay====
 +
''by Sean''
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/ protocol]
-
==D- Lemon scent==
+
===Lemon scent===
-
===Bacterial culture===
+
====Bacterial culture====
''by Laetitia''
''by Laetitia''
 +
[[File:20 08 bacterial culture.jpg| 500px|right]]
Here are the results of the bacterial culture of yesterday
Here are the results of the bacterial culture of yesterday
-
[[File:20 08 bacterial culture.jpg| 500px]]
 
Bacteria with the pGEMTeasy without the DNA insert are blue and the bacteria with the pGEMTeasy and the DNA insert (PS, GS or LS gene) are white
Bacteria with the pGEMTeasy without the DNA insert are blue and the bacteria with the pGEMTeasy and the DNA insert (PS, GS or LS gene) are white
Line 27: Line 115:
Preculture 20ml of DH5a pPSI (apra 1/2000)
Preculture 20ml of DH5a pPSI (apra 1/2000)
 +
====CAD PCR====
 +
Today we received our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)
-
===CAD PCR===
+
so we do a PCR (with GoTaq):
-
Today we have receive our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)
+
-
so we do a PCR (with go taq):
+
{| class="wikitable centre" width="50%"
 +
|+ PCR of CAD
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|DNA of CAD
 +
|"very small amount"
 +
|-
 +
|GoTaq buffer 5X
 +
|10μl
 +
|-
 +
|DMSO
 +
|1.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|23.25µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|4µl
 +
|-
 +
|dNTP
 +
|1µl
 +
|-
 +
|iPS79
 +
|5µl
 +
|-
 +
|iPS80
 +
|5µl
 +
|-
 +
|GoTaq polymerase
 +
|.25µl
 +
|}
-
23.25 µl --> H2O
+
{| class="wikitable centre" width="50%"
 +
|+ PCR cycle
 +
|-
 +
! scope=col | step
 +
! scope=col | temperature (°C)
 +
! scope=col | time (min)
 +
|-
 +
|1
 +
|95
 +
|2
 +
|-
 +
|2
 +
|95
 +
|.5
 +
|-
 +
|3
 +
|58
 +
|.5
 +
|-
 +
|4
 +
|72
 +
|1.75
 +
|-
 +
|5
 +
|72
 +
|5
 +
|}
-
10 µl --> 5xbuffer
+
====CAD cloning in pGEMTeasy====
 +
''by Sean''
-
1 µl --> dNTP
+
[https://2014.igem.org/Team:Paris_Saclay/Protocols/ protocol]
-
4 µl --> MgCl2
+
==== Electrophoresis of pPSI digested by PacI ====
-
 
+
''by Eugene''
-
5 µl iPS 79 (Primer)
+
-
 
+
-
5 µl iPS 80
+
-
 
+
-
Very small quantity of DNA
+
-
 
+
-
1.5 µl --> DMSO
+
-
0.25 µl --> Gotaq
+
We made an electrophoresis of pPSI to verify if the digestion made [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/19#Digestion_of_PCR_results 19th August] has worked. It is useful before starting the purification of the plasmid.
-
PCR cycle
+
#non-digested pPSI 3µl
 +
#digested pPSI 3µl
 +
#ladder 10µl
-
95°  2 min
+
[[File:Paris_Saclay_140820.jpg|center]]
-
95°  30 sec
+
-
58°  30 sec
+
-
72°  1 min 45
+
-
72°  5 min
+
-
===Migration of pPS1 digested by PacI===
+
====Migration of pPS1 digested by PacI for the purification====
''by Huang vu and Laetitia''
''by Huang vu and Laetitia''
Line 72: Line 211:
The totality of the sample is filled (around 38µL) and we filled 20µl of ladder. Migration 100V  
The totality of the sample is filled (around 38µL) and we filled 20µl of ladder. Migration 100V  
-
After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf, after the purification
+
After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification
-
 
+
====Digestion of CAD and PMCS5 at 37°C====
-
=== Electrophoresis of pPSI ===
+
-
''by Eugene''
+
-
 
+
-
We make an electrophoresis of pPSI to verify if the digestion made 19/08 has worked. It is usefull before starting the purification of the plasmid.
+
-
 
+
-
=== Digestion of CAD and PMCS5 ===  
+
''by Eugene''
''by Eugene''
Line 122: Line 255:
|}
|}
 +
==Photo of the Day==
 +
[[File:Paris Saclay 20_august.jpg|300px|center]]
 +
'''Members present:'''
 +
* Instructors and advisors: Alice, Solenne and Sylvie.
 +
* Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.
-
====B - Construction of the fusion protein (color)=====
+
{{Team:Paris_Saclay/notebook_footer}}
-
PCR with Gotaq
+
-
 
+
-
22.75 µl --> H2O
+
-
 
+
-
10 µl --> 5xbuffer
+
-
 
+
-
1 µl --> dNTP
+
-
 
+
-
4 µl --> MgCl2
+
-
 
+
-
5 µl iPS 83 (Primer)
+
-
 
+
-
5 µl iPS 84
+
-
 
+
-
0.5 µl --> DNA
+
-
 
+
-
1.5 µl --> DMSO
+
-
 
+
-
0.25 µl --> Gotaq
+
-
 
+
-
 
+
-
PCR cycle
+
-
 
+
-
95°  2 min
+
-
95°  30 sec
+
-
58°  30 sec
+
-
72°  1 min 45
+
-
72°  5 min
+

Latest revision as of 15:08, 14 October 2014

Contents

Wednesday 20th August

Lab work

Petri dishes

by Laetitia

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

Construction of the fusion protein (color)

PCR

PCR of chromoprotein
components volumes
DNA .5µl
GoTaq buffer 5X 10μl
DMSO 1.5µl
H2O 22.75µl
MgCl2 4µl
dNTP 1µl
iPS83 5µl
iPS84 5µl
GoTaq polymerase .25µl
PCR cycle
step temperature (°C) time (min)
1 95 2
2 95 .5
3 58 .5
4 72 1.75
5 72 5

Cloning in pGEMTesay

by Sean

protocol

Lemon scent

Bacterial culture

by Laetitia

20 08 bacterial culture.jpg

Here are the results of the bacterial culture of yesterday


Bacteria with the pGEMTeasy without the DNA insert are blue and the bacteria with the pGEMTeasy and the DNA insert (PS, GS or LS gene) are white

Then, we have chosen 3 white colonies for each DNA insert and we have launched 3 corresponding liquid cultures :

- 5mL LB + ampiciline (1/1000e)

- 1 white colony

The 9 cultures are incubated at 37°C


by Melanie

Preculture 20ml of DH5a pPSI (apra 1/2000)

CAD PCR

Today we received our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)

so we do a PCR (with GoTaq):

PCR of CAD
components volumes
DNA of CAD "very small amount"
GoTaq buffer 5X 10μl
DMSO 1.5µl
H2O 23.25µl
MgCl2 4µl
dNTP 1µl
iPS79 5µl
iPS80 5µl
GoTaq polymerase .25µl
PCR cycle
step temperature (°C) time (min)
1 95 2
2 95 .5
3 58 .5
4 72 1.75
5 72 5

CAD cloning in pGEMTeasy

by Sean

protocol

Electrophoresis of pPSI digested by PacI

by Eugene

We made an electrophoresis of pPSI to verify if the digestion made 19th August has worked. It is useful before starting the purification of the plasmid.

  1. non-digested pPSI 3µl
  2. digested pPSI 3µl
  3. ladder 10µl
Paris Saclay 140820.jpg

Migration of pPS1 digested by PacI for the purification

by Huang vu and Laetitia

pPS1 previously digested by PacI is filled inside an agarose gel :

-1g agarose

-1mL TAE

-2 µL BET

The totality of the sample is filled (around 38µL) and we filled 20µl of ladder. Migration 100V

After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification

Digestion of CAD and PMCS5 at 37°C

by Eugene

components volumes
CAD 5 μl
10x buffer fastDigest 1 μl
PacI 0.5 µl
H2O 3.5 µl


components volumes
PMCS5 15 μl
10x buffer fastDigest 2 μl
PacI 0.5 µl
H2O 2.5 µl

Photo of the Day

300px

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/20"