Team:Freiburg/Notebook/16 August

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{{:Team:Freiburg/Templates/header}}
{{:Team:Freiburg/Templates/header}}
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<div id="notebook-page-header">
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<div id="notebook-back" width="100px" >
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/15_August">Previous entry</a>
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</div>
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<div id="notebook-title">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 16 August </a>
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</div>
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<div id="notebook-next">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/17_August">Next entry</a>
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</div>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
Line 11: Line 25:
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Picking clones===
-
'''Investigators:NAME'''
+
'''Investigators: Sandra'''
 +
Picking clones of transformed cells with LovTAp, NotGate, pSB1T3.
 +
==<span style="color:red;">red light receptor</span>==
 +
===continuing with PCR of cph8===
-
==<span style="color:red;">red light receptor</span>==
+
'''Investigators:Julia'''
 +
<br/>
 +
2µl of the PCR reaction from 15th Aug. were loaded onto a gel.<br/>
 +
But there was no band. Jakob is going to repeat the PCR.<br/>
-
===NAME OF YOUR EXPERIMENT===
+
===3a-assembly with promotor+ribosome binding site infront of pcyA and ho1===
-
'''Investigators:NAME'''
+
'''Investigators:Julia'''
 +
'''Digestion'''
 +
*PR1 in pSB1C3
 +
*PR2 in pSB1C3
 +
*pcyA (pSB1T3)
 +
#pcyA+terminator(d)in pSB1T3
 +
*ho1+Terminator (pSB1T3)
 +
*vector (pSB1K3)
 +
 +
Amount of DNA and H20:
 +
 +
{| cellpadding="10" cellspacing="0" border="1"
 +
|Sample
 +
|500(ng)/DNA concentration (ng/μl)
 +
|H20 μl
 +
|-
 +
|pcyATd
 +
|2.8
 +
|35.2
 +
|-
 +
|pcyaTb
 +
|2.9
 +
|35.1
 +
|-
 +
|PR2
 +
|7.2
 +
|30.8
 +
|-
 +
|PR1
 +
|6.1
 +
|31.9
 +
|-
 +
|S22a
 +
|2,5
 +
|35.5
 +
|-
 +
|pSB1K3
 +
|20
 +
|18
 +
|}
 +
 +
 +
Enzymes necessary for digestion:
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|
 +
|PR1/PR2
 +
|pcyA/S22a
 +
|vector
 +
|-
 +
|enzyme 1
 +
|EcoRI
 +
|XbaI
 +
|EcoRI
 +
|-
 +
|enzyme 2
 +
|SpeI
 +
|PstI
 +
|PstI
 +
|}
 +
 +
 +
-incubated for 1 hours at 37°C.<br/>
 +
-heated for 20 minutes at 80°C
 +
 +
 +
'''Ligation'''
 +
 +
Ligation of:
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|
 +
|PcyATd
 +
 +
|S22a (ho1)
 +
|-
 +
|PR1
 +
|'''1 pTd'''
 +
|'''1 pTb'''
 +
 +
|-
 +
|PR2
 +
|'''2 pTd'''
 +
|'''2 pTb'''
 +
 +
|}
 +
 +
===PCR===
 +
 +
'''Investigator: Jakob'''
 +
 +
 +
'''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:
 +
 +
Jakob
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
 +
 +
16.08.2011
 +
 +
|-
 +
| 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: 12.08.2011
 +
 +
Order primers
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
 +
 +
Red light receptor, amplify Cph8
 +
 +
|}
 +
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;"| P 58 (1:10)
 +
 +
|-
 +
| 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;"| P 59 (1:10)
 +
 +
|-
 +
| 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;"| JT122 5ng/µl
 +
 +
|-
 +
| 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;"|
 +
 +
|}
 +
The program we used:
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>Temp.</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>Time</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Nr. of cycles
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>5´</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1x</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center>
 +
|rowspan="3" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>10x</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>65°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1´5´´</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center>
 +
| rowspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>20x</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1´5´´</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>7´</center>
 +
| rowspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1x</center>
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>4°</center>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>∞</center>
 +
|}
 +
<br/>
 +
'''We got the information for the program and the primer sequences from the iGEM Team Uppsala Sweden.'''
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===2A Assembly like August 15===
-
'''Investigators:NAME'''
+
'''Investigators:Theo'''
 +
 
 +
 
 +
This time I used all the amount of S15 plasmid DNA we had, did an overnight restriction and run the gel.<br>
 +
[[File:Freiburg11 1608gelextraction.png]]
 +
<br>
 +
extraction as per SOP and ligation with S4 (cut with SpeI, PstI), Transformation in competent cells.
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==

Latest revision as of 01:09, 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

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

Picking clones

Investigators: Sandra

Picking clones of transformed cells with LovTAp, NotGate, pSB1T3.

red light receptor

continuing with PCR of cph8

Investigators:Julia
2µl of the PCR reaction from 15th Aug. were loaded onto a gel.
But there was no band. Jakob is going to repeat the PCR.

3a-assembly with promotor+ribosome binding site infront of pcyA and ho1

Investigators:Julia

Digestion

  • PR1 in pSB1C3
  • PR2 in pSB1C3
  • pcyA (pSB1T3)
  1. pcyA+terminator(d)in pSB1T3
  • ho1+Terminator (pSB1T3)
  • vector (pSB1K3)


Amount of DNA and H20:

Sample 500(ng)/DNA concentration (ng/μl) H20 μl
pcyATd 2.8 35.2
pcyaTb 2.9 35.1
PR2 7.2 30.8
PR1 6.1 31.9
S22a 2,5 35.5
pSB1K3 20 18


Enzymes necessary for digestion:

PR1/PR2 pcyA/S22a vector
enzyme 1 EcoRI XbaI EcoRI
enzyme 2 SpeI PstI PstI


-incubated for 1 hours at 37°C.
-heated for 20 minutes at 80°C


Ligation

Ligation of:

PcyATd S22a (ho1)
PR1 1 pTd 1 pTb
PR2 2 pTd 2 pTb

PCR

Investigator: Jakob


PCR


Name:

Jakob

Date:

16.08.2011

Continue from Experiment: 12.08.2011

Order primers

Project Name:

Red light receptor, amplify Cph8

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 58 (1:10)
2.5µl Primer dw P 59 (1:10)
1µl dNTPs of Template DNA
1µl DNA-Template JT122 5ng/µl
0.5 µl Phusion (add in the end)

The program we used:


Temp.
Time
 Nr. of cycles
98°
5´
1x
98°
30´´
10x
65°
30´´
72°
1´5´´
98°
30´´
20x
72°
1´5´´
72°
7´
1x
4°
∞


We got the information for the program and the primer sequences from the iGEM Team Uppsala Sweden.

Lysis cassette

2A Assembly like August 15

Investigators:Theo


This time I used all the amount of S15 plasmid DNA we had, did an overnight restriction and run the gel.
Freiburg11 1608gelextraction.png


extraction as per SOP and ligation with S4 (cut with SpeI, PstI), Transformation in competent cells.

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/16_August"