Team:Freiburg/Notebook/11 August

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 11 August </a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Skypephoned with the iGEM-Team Uppsala===
-
'''Investigators:NAME'''
+
'''Investigators:Theo, Rüdiger, Jakob'''
 +
We talked to two guys (Lei Sun and Hamid Gavali) from the igem team uppsala.
 +
They gave us some informations and support, quite nice guys.
 +
Informations:
 +
* few Primer sequences
 +
* Touchdown-pcr protocol
 +
 +
Thx a lot for that!!!
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===PCR===
-
'''Investigators: Sophie'''
+
 
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Sophie
 +
 
 +
 
 +
 
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 11.8.11
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: new experiment
 +
 
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: changes in the LovTAP for 3A assembly
 +
 
 +
|}
 +
PCR-Mixture for one Reaction:
 +
 
 +
For a 50 µl reaction use
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
What program do you use? Normal PCR with 60°C annealing temperature
 +
 
 +
 
 +
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
 +
 
 +
 
 +
How did you label the PCR-Product, where is it stored and what do you do next?
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
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==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
===NAME OF YOUR EXPERIMENT: Tramsformation with inducible Promoter===
+
===Tramsformation with inducible Promoter===
-
===='''Investigators: Sophie'''====
+
'''Investigators: Sophie'''
We didn't get any clones with both Plastic binding domain and Promoter-RBS inside, the Pbd seems to be toxic when expressed.So we want to try if it is possible to get cells with an inducible Promoter and Pbd. In this case we could cultivate the cells and induce expression of the Pbd only shortly before lysis. As our Light-inducible Promoters are not finished we use the IPTG inducible Promoter BBa_J04500 (Plate4, 12A)
We didn't get any clones with both Plastic binding domain and Promoter-RBS inside, the Pbd seems to be toxic when expressed.So we want to try if it is possible to get cells with an inducible Promoter and Pbd. In this case we could cultivate the cells and induce expression of the Pbd only shortly before lysis. As our Light-inducible Promoters are not finished we use the IPTG inducible Promoter BBa_J04500 (Plate4, 12A)
 +
 +
===PCR===
 +
 +
'''Investigators: Rüdiger'''
 +
 +
PCR of precipitator.
 +
 +
Primer fw:
 +
*p51
 +
*p52
 +
*p53
 +
 +
Primer dw:
 +
*p54
 +
 +
DNA template: S43
 +
 +
DMSO was used to reduce Tm about 5°C.
 +
 +
[[File:Freiburg2011_tesdigestRüdi.jpg|600px]]
 +
 +
For the first a51, a52 and a53 6% DMSO was used.
 +
For b51, b52 and b53 9% Dmso was used.
 +
For c51, c52 and c53 12% Dmso was used.
 +
 +
===Picking colonies===
 +
 +
'''Investigators: Rüdiger'''
 +
 +
5 colonies were picked and incubated overnight in LB amp medium.
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 01:07, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!


Contents

green light receptor

Skypephoned with the iGEM-Team Uppsala

Investigators:Theo, Rüdiger, Jakob

We talked to two guys (Lei Sun and Hamid Gavali) from the igem team uppsala. They gave us some informations and support, quite nice guys.

Informations:

  • few Primer sequences
  • Touchdown-pcr protocol

Thx a lot for that!!!

blue light receptor

PCR

Name: Sophie


Date: 11.8.11
Continue from Experiment: new experiment


Project Name: changes in the LovTAP for 3A assembly

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 5x Phusion Buffer of Primer
2.5µl Primer fw
2.5µl Primer dw
1µl dNTPs of Template DNA
1µl DNA-Template
0.5 µl Phusion (add in the end)

What program do you use? Normal PCR with 60°C annealing temperature


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.


How did you label the PCR-Product, where is it stored and what do you do next?

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

Tramsformation with inducible Promoter

Investigators: Sophie We didn't get any clones with both Plastic binding domain and Promoter-RBS inside, the Pbd seems to be toxic when expressed.So we want to try if it is possible to get cells with an inducible Promoter and Pbd. In this case we could cultivate the cells and induce expression of the Pbd only shortly before lysis. As our Light-inducible Promoters are not finished we use the IPTG inducible Promoter BBa_J04500 (Plate4, 12A)

PCR

Investigators: Rüdiger

PCR of precipitator.

Primer fw:

  • p51
  • p52
  • p53

Primer dw:

  • p54

DNA template: S43

DMSO was used to reduce Tm about 5°C.

Freiburg2011 tesdigestRüdi.jpg

For the first a51, a52 and a53 6% DMSO was used. For b51, b52 and b53 9% Dmso was used. For c51, c52 and c53 12% Dmso was used.

Picking colonies

Investigators: Rüdiger

5 colonies were picked and incubated overnight in LB amp medium.

                       

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