Team:Freiburg/Notebook/24 August

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 24 August </a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===Digest===
+
===Digest of CcaR===
'''Investigators: Jakob'''
'''Investigators: Jakob'''
Line 14: Line 29:
Digest for 2h at 37°C
Digest for 2h at 37°C
-
===Transformation===
+
===Transformation of CcaR===
'''Investigators: Jakob''' <br/>
'''Investigators: Jakob''' <br/>
-
 
===Testdigest of quickchanged CcaS===
===Testdigest of quickchanged CcaS===
Line 105: Line 119:
What program do you use?
What program do you use?
-
20 cycles with an annealing temperature of 65/68/71 ºC (gradient-PCR) then 10 cycles with an annealing temperature of 65/70/75 ºC
+
20 cycles with an annealing temperature of 65/68/71 ºC (gradient-PCR) and touchdown then 10 cycles with an annealing temperature of 65/70/75 ºC and touchdown
 +
<br/>
 +
digested with DpnI
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
 +
[[File:Freiburg_blue_24.08..jpg]]
-
 
+
How did you label the PCR-Product, where is it stored and what do you do next?<br/>
-
How did you label the PCR-Product, where is it stored and what do you do next?
+
the PCR-products are labelled Not, nOt and noT and are stored in the "unverdaute Minipreps II"-box (or in the Gibson box?) If Sequencing is correct, I will do a new 3A assembly with the LovTAP-PCR product and an Amp-vector next.
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
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===NAME OF YOUR EXPERIMENT===
===NAME OF YOUR EXPERIMENT===
-
'''Investigators: NAME'''
+
===PCR===
 +
 
 +
 
 +
{| 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:Rüdiger
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 +
 
 +
 
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 24.08
 +
 
 +
|-
 +
| 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 (Date)-
 +
 
 +
(Name)
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Precipitator
 +
 
 +
|}
 +
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;"| P68Primer
 +
 
 +
|-
 +
| 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;"| P70Primer
 +
 
 +
|-
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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
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNAPrimer
 +
 
 +
|-
 +
| 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;"| GST-6-P-1 a,b,c,d,ePrimer
 +
 
 +
|-
 +
| 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?
 +
 
 +
Annealing 60-70
 +
 
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 01:11, 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

Digest of CcaR

Investigators: Jakob

  • 10µl BSA
  • 10µl NEB
  • 2µl DpnI
  • 38µl DNA

Digest for 2h at 37°C

Transformation of CcaR

Investigators: Jakob

Testdigest of quickchanged CcaS

Investigators: Julia

today I prepared 9 minipreps of the the quickchanged CcaS.
They were digested with EcoRI and loaded on a gel.
Those which are not cut twice were changed during PCR correctly.
We will send them for sequencing today.

Freiburg 8 25 2011 qS50 S51.Jpg
gel loaded with: Ladder 1 kb, qS50 a,b,c,d , qS51 a, b, c, d
lower lane:Ladder 1 kb qS51 e

positive clones are qS50b and qS51a.
concentration of minpreps: qS50b: 88,6 ng/μl and qS51a: 81,1 ng/μl.

blue light receptor

Transformation

Investigators: Sandra

Repeat of the transformation from yesterday with the Lovtap-NotGate-pSB1A3 vector.

PCR

Name: Sophie


Date: 24.08.11
Continue from Experiment: primer design (Date) 19.08.11

(Name) Sandra

Project Name: Blue light

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 P24
2.5µl Primer dw P79
1µl dNTPs of Template DNA
1µl DNA-Template M45
0.5 µl Phusion (add in the end)

What program do you use?

20 cycles with an annealing temperature of 65/68/71 ºC (gradient-PCR) and touchdown then 10 cycles with an annealing temperature of 65/70/75 ºC and touchdown
digested with DpnI


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel. Freiburg blue 24.08..jpg

How did you label the PCR-Product, where is it stored and what do you do next?
the PCR-products are labelled Not, nOt and noT and are stored in the "unverdaute Minipreps II"-box (or in the Gibson box?) If Sequencing is correct, I will do a new 3A assembly with the LovTAP-PCR product and an Amp-vector next.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

NAME OF YOUR EXPERIMENT

PCR

Name:Rüdiger


Date: 24.08
Continue from Experiment (Date)-

(Name)

Project Name: Precipitator

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 P68Primer
2.5µl Primer dw P70Primer
1µl dNTPs of Template DNAPrimer
1µl DNA-Template GST-6-P-1 a,b,c,d,ePrimer
0.5 µl Phusion (add in the end)

What program do you use?

Annealing 60-70

                       

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