Team:Freiburg/Notebook/29 August

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 29 August </a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===PCR to amplify Green light Promotor PcpcG===
+
==='''PCR to amplify Green light Promotor PcpcG'''===
'''Investigators:Julia'''
'''Investigators:Julia'''
Line 71: Line 85:
&nbsp;
&nbsp;
-
[[File:Freiburg11_8_31_2011.Jpg‎]]
+
[[File:Freiburg11_8_31_2011.Jpg‎|350px|350px]]
<br/>
<br/>
the lane labeled P, is the PCR product.Expected size: 264 bp
the lane labeled P, is the PCR product.Expected size: 264 bp
-
===Testdigest of quickchanged CcaR and Precipitator===
+
==='''Testdigest of quickchanged CcaR and Precipitator'''===
'''Investigators:Julia,with help of the team'''
'''Investigators:Julia,with help of the team'''
Line 225: Line 239:
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
 +
 +
===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
 +
 +
 +
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 29.08
 +
 +
|-
 +
| 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 PCR Rüdiger (Date)-22.08.
 +
 +
(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
 +
 +
|-
 +
| 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 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
 +
 +
 +
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?
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border-top:0.0007in solid #000000;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Name
 +
| style="border-top:0.0007in solid #000000;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <nowiki>#</nowiki>
 +
| style="border-top:0.0007in solid #000000;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Template
 +
| style="border-top:0.0007in solid #000000;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Primer
 +
| style="border:0.0007in solid #000000;padding:0.0382in;"| Anneal. Temp.
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| For David Vector
 +
 +
Bam H1 +EcoR1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 3
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 87+88
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 65
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 2
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 4_2
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 91+92
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 65
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 3
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 4
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 98+90
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 65
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Gibson
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 4
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| pGEX-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 85+44
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 66
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 5
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| pGEX-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 85+45
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 66
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 6
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| pGEX-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 85+46
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 66
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Registry cloning
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 7
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| pGEX-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 85+48
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 66
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 8
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| pGEX-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 48+84
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 66
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Toolbox Gibson>GST-6-p-1
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 9
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 2
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 81+73
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 67
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 10
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 4_2
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 82+71
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 67
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 11
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Precipitator 4
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 83+72
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 67
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Control
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 12
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Lambda DNA
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Primers
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"|
 +
 +
|}
 +
 +
===Testdigest of Precipitator in iGEM Vektor pSB1C3===
===Testdigest of Precipitator in iGEM Vektor pSB1C3===

Latest revision as of 01:13, 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

PCR to amplify Green light Promotor PcpcG

Investigators:Julia PCR


Name: Julia

 

Date: 29.08.2011
Project Name: Amplification of Green light Promotor

 

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20
10µl 5x Phusion Buffer
2.5µl Primer     fw  tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC
2.5µl Primer     dw  tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC
1µl dNTPs
1µl DNA-Template  original Synechocystis genome
0.5 µl Phusion  

 

What program do you use?

 

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

  Freiburg11 8 31 2011.Jpg

the lane labeled P, is the PCR product.Expected size: 264 bp

Testdigest of quickchanged CcaR and Precipitator

Investigators:Julia,with help of the team


3μl of the Miniprep were digested with EcoRI and PstI.
Freiburg11-08-29 Testdigest.jpeg


we are going to sequence the Precipitator samples 61a and b
(wrong labeling on the pic, its the 63 in the right corner)
62 b and f
63 c and d
and of the green light response regulator qCcaR 17b and 18a

blue light receptor

3A-assembly

Investigators: Sandra

Digestion I:

  • Lovtap
  • Not-Gate
  • pSB1A3
  • pSB1T3

stored in the blue light box.

Ligation

  • ♥-A3 Not
  • ♥-A3 nOt
  • ♥-A3 noT
  • ♥-T3 Not
  • ♥-T3 nOt
  • ♥-T3 noT

stored in the ligation box.

Digestion II:

Also digestion with new amplified vector

  • pSB1C3

Lovtap was digested again this time also with DpnI. So for this ligation the ♥-PCR3A, digested with DpnI, and pSB1C3 (new amplified with PCR, already digested with DpnI) and Not, nOt, noT were used.

  • ♥-C3 Not
  • ♥-C3 nOt
  • ♥-C3 noT

Transformation

Investigators: Sophie

transformation of:

  • ♥-A3 Not
  • ♥-A3 nOt
  • ♥-A3 noT
  • ♥-T3 Not
  • ♥-T3 nOt
  • ♥-T3 noT

Incubation over night at 37°C.

PCR

Investigator: Sophie


PCR


Name:


Date:


Continue from Experiment: new


Project Name:

more NOT-gate with mutated restriction site (NHE I instead of Spe I)

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 P 24 (1:10)
2.5µl Primer dw P 79 (1:10)
1µl dNTPs of Template DNA
1µl DNA-Template

M45 (Bba_Q04400)

0.5 µl Phusion (add in the end)

The program we used: RT 2step (see last PCR of NOT-gate)
Freiburg11.08.11.jpg

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

PCR

Name:Rüdiger


Date: 29.08
Continue from Experiment PCR Rüdiger (Date)-22.08.

(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


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?


Name # Template Primer Anneal. Temp.
For David Vector

Bam H1 +EcoR1

1 Precipitator 3 87+88 65
2 Precipitator 4_2 91+92 65
3 Precipitator 4 98+90 65
Gibson 4 pGEX-6-p-1 85+44 66
5 pGEX-6-p-1 85+45 66
6 pGEX-6-p-1 85+46 66
Registry cloning 7 pGEX-6-p-1 85+48 66
8 pGEX-6-p-1 48+84 66
Toolbox Gibson>GST-6-p-1 9 Precipitator 2 81+73 67
10 Precipitator 4_2 82+71 67
11 Precipitator 4 83+72 67
Control 12 Lambda DNA Primers


Testdigest of Precipitator in iGEM Vektor pSB1C3

Investigators: Julia and others

results see above under "green light receptor"

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/29_August"