Team:Freiburg/Notebook/9 August

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 9 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===
+
===Results Transformation===
-
'''Investigators:NAME'''
+
We dont see any colonies
 +
*To-do: do quickchange again.
 +
 +
===Quickchange===
 +
<br/>
 +
'''Investigator:Jakob'''
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 +
'''PCR'''
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 +
 +
{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name:
 +
 +
Jakob
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
 +
 +
09.08.2011
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 +
|-
 +
| 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:
 +
 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
 +
 +
Quickchange CcaR and CcaS
 +
 +
|}
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PCR-Mixture for one Reaction:
 +
 +
For a 50 µl reaction use
 +
 +
 +
{| style="border-spacing:0;"
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| 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
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
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| 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;"|
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer: P12(CcaR) and P14(CcaS
 +
 +
|-
 +
| 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;"| Primer: P13(CcaR) and P15(CcaS)
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA: CcaR and CcaS
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
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| 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)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
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|}
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 +
 +
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*'''Digest with DpnI'''
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<br/>
 +
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*'''Transformation'''
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<br/>
 +
 +
* And again, I'm such a fool...
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
Line 29: Line 121:
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Lysis cassette sequencing===
-
'''Investigators:NAME'''
+
'''Investigators:Theo'''
 +
Sequencing showed part of the E.coli genome inside the restriction sites where the lysis cassette should be (aaargh)
 +
<br>
 +
[[File:Lys1-P8.gb]]
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===new 3A assenbly with Amp Vector===
-
new 3A assenbly with Amp Vector
+
'''Investigators: Sophie'''
'''Investigators: Sophie'''
====3A assembly====
====3A assembly====
-
The last cloning of Theo and Jakob produced one clone with GFP-plastic-binding-domain (GFP-pbd) inside but without Promoter-Ribosome-binding site (PR). So I tried again to combine the GFP-pbd with PRs via 3a assenbly. I used two different PRs (4 and 6) with different RBS binding strenght.
+
The last cloning of Theo and Jakob produced one clone with GFP-plastic-binding-domain (GFP-pbd) inside but without Promoter-Ribosome-binding site (PR). So I tried again to combine the GFP-pbd with PRs via 3a assenbly. I used two different PRs (PR4 and PR6) with different RBS binding strenght.
Name of the samples:
Name of the samples:
ε5 (GFP-pbd),
ε5 (GFP-pbd),

Latest revision as of 01:06, 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

Results Transformation

We dont see any colonies

  • To-do: do quickchange again.


Quickchange


Investigator:Jakob

PCR


Name:

Jakob

Date:

09.08.2011

Continue from Experiment:
Project Name:

Quickchange CcaR and CcaS

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20
10µl 5x Phusion Buffer
2.5µl Primer fw Primer: P12(CcaR) and P14(CcaS
2.5µl Primer dw Primer: P13(CcaR) and P15(CcaS)
1µl dNTPs
1µl DNA-Template DNA: CcaR and CcaS
0.5 µl Phusion (add in the end)



  • Digest with DpnI


  • Transformation


  • And again, I'm such a fool...

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

Lysis cassette sequencing

Investigators:Theo


Sequencing showed part of the E.coli genome inside the restriction sites where the lysis cassette should be (aaargh)
File:Lys1-P8.gb

Precipitator

new 3A assenbly with Amp Vector

Investigators: Sophie

3A assembly

The last cloning of Theo and Jakob produced one clone with GFP-plastic-binding-domain (GFP-pbd) inside but without Promoter-Ribosome-binding site (PR). So I tried again to combine the GFP-pbd with PRs via 3a assenbly. I used two different PRs (PR4 and PR6) with different RBS binding strenght. Name of the samples: ε5 (GFP-pbd), pSB1A3, PR4, PR6. stored in "Minipreps, verdaut"-box

Ligation

I ligated ε5 (GFP-pbd) to PR4 or PR6 and to the Amp-Vector. Therefore I once took the ratio 3:1 insert to vector and once the ration 1:1 insert to vector.

                       

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