Team:Freiburg/Notebook/18 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/17_August">Previous entry</a>
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<div id="notebook-title">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 18 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/19_August">Next entry</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===
+
===Quickchange===
-
 
+
-
'''Investigators:NAME'''
+
-
 
+
 +
'''Investigators:Jakob'''
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
Line 20: Line 32:
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
picked colonies from yesterdays trnasformation. <br/>
-
 
+
<br/>
-
'''Investigators:NAME'''
+
new PCR in order to again cph8 from pJT122.<br/>last time PCR did not work.
-
 
+
-
 
+
-
 
+
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===2A assembly===
-
'''Investigators:NAME'''
+
'''Investigators:Theo'''
 +
Sent for sequencing
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
Line 165: Line 175:
Add 1 μl Loading dye buffer and load the gel.
Add 1 μl Loading dye buffer and load the gel.
-
Take a picture of the gel, print picture and label the lanes!  
+
Take a picture of the gel, print picture and label the lanes! <br/>
 +
[[File:Freigem18.8.11.jpg]]
 +
<br/>
 +
===3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector===
 +
<br/>
 +
 +
Investigators: Sophie
 +
 +
'''Digestion'''
 +
 +
Amount of DNA and H20:
 +
 +
{| cellpadding="10" cellspacing="0" border="1"
 +
|Sample
 +
|DNA μl
 +
|H20 μl
 +
|-
 +
|IPTG-Promoter
 +
|2,5
 +
|33
 +
|-
 +
|GFP-pbd
 +
|3
 +
|36
 +
|-
 +
|pSB1A3
 +
|20
 +
|18
 +
|}
 +
 +
 +
Enzymes necessary for digestion:
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|
 +
|GFP-pbd: 4 4 4 or 6 6 8
 +
|IPTG-Promoter: S54
 +
|vector: psB1C3
 +
|-
 +
|enzyme 1
 +
|EcoRI
 +
|XbaI
 +
|EcoRI
 +
|-
 +
|enzyme 2
 +
|NheI
 +
|PstI
 +
|PstI
 +
|}
 +
 +
 +
*Incubation at 37°C for 6 hours
 +
*20 minutes at 80°C
 +
<br/>
 +
<br/>
 +
 +
===Ligation===
 +
 +
 +
{| 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: 18.08.11
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date: 18.08.11 Name: Sophie
 +
 +
Experiment: Cloning
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: inducible Promoter for pbd with Cm-vector
 +
 +
|}
 +
'''Procedure'''
 +
 +
 +
PCR tube:
 +
 +
total volume 20 μl
 +
 +
 +
# add H<sub>2</sub>O (17 μl -X-Y-Z)
 +
# add 2 μl Ligase Buffer 10x
 +
# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
 +
# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
 +
# Add 1 μl T4-DNA Ligase
 +
# Incubate 10-30 min at room temperature
 +
# heat for 20 minutes at 80°C
 +
# store at -20°C or directly proceed to transformation
 +
 +
 +
{| style="border-spacing:0;"
 +
| 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;"| Name of part
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ratio Insert:Vector
 +
 +
<nowiki>= 3:1 or 1:1</nowiki>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Volume (μl)
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| X insert 1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S54
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| both
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2 or 3
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Y insert 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PR 444 bzw. PR 668
 +
| 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 or 3
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Z vector
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Psb1A3
 +
| 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 or 1
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O
 +
| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="background-color:#bfbfbf;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;"| 11
 +
 +
|}
 +
'''Documentation:'''
 +
 +
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Stored in Ligation-box, single parts stored in minipreps, verdaut-box
 +
 +
name of parts see above.
 +
 +
|}
 +
 +
===PCR===
 +
 +
'''Investigators: Rüdiger'''
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|name
 +
|templates
 +
|primer
 +
|-
 +
|a
 +
|pGex-6-P-1a
 +
|P62+P63
 +
|-
 +
|b
 +
|pGex-6-P-1b
 +
|´´
 +
|-
 +
|a10
 +
|pGex-G-P-1a
 +
|´´
 +
|-
 +
|b10
 +
|pGex-6-P-1b
 +
|´´
 +
|}
 +
PCR did not work. New primer design.
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

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

Quickchange

Investigators:Jakob

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



red light receptor

picked colonies from yesterdays trnasformation.

new PCR in order to again cph8 from pJT122.
last time PCR did not work.

Lysis cassette

2A assembly

Investigators:Theo


Sent for sequencing

Precipitator

Miniprep

zymo Kit


Name:Sophie


Date:18.08.11
Continue from Experiment: Cloning: ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too. (Date): 12.08.11

(Name): Sophie

Project Name: inducible Promoter with pbd with cm-vector


Documentation:

Why are you doing this experiment? Name the parts which you extract.

ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too.

Name of the parts: GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2


Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.


GFP-pbd 4 4 4: 164,1 ng/µl

GFP-pbd 4 1 3: 100,8 ng/µl

GFP-pbd 6 6 8: 163,5 ng/µl

GFP-pbd 4 2 2: 144,4 ng/µl

How did you label your probes and where are they stored?


Labelled GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2

stored in -20 until sequencing


Testdigest

Name: Sophie


Date: 18.08.11
Continue from Experiment: Miniprep (Date): 18.08.11

(Name): Sophie

Project Name:inducible promoter for pbd with cm-vector

For one reaction you need: For Mastermix: Number of samples+2extra


4μl H2O 20
1μl Buffer, NEB4 5
1μl BSA (10x) 5
0,5 μl Enzym 1 2,5
0,5 μl Enzym 2 2,5
3 μl DNA

10 μl total volume


Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.


Add 1 μl Loading dye buffer and load the gel.

Take a picture of the gel, print picture and label the lanes!
Freigem18.8.11.jpg

3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector


Investigators: Sophie

Digestion

Amount of DNA and H20:

Sample DNA μl H20 μl
IPTG-Promoter 2,5 33
GFP-pbd 3 36
pSB1A3 20 18


Enzymes necessary for digestion:

GFP-pbd: 4 4 4 or 6 6 8 IPTG-Promoter: S54 vector: psB1C3
enzyme 1 EcoRI XbaI EcoRI
enzyme 2 NheI PstI PstI


  • Incubation at 37°C for 6 hours
  • 20 minutes at 80°C



Ligation

Name: Sophie Date: 18.08.11
Continue from Date: 18.08.11 Name: Sophie

Experiment: Cloning

Project Name: inducible Promoter for pbd with Cm-vector

Procedure


PCR tube:

total volume 20 μl


  1. add H2O (17 μl -X-Y-Z)
  2. add 2 μl Ligase Buffer 10x
  3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
  4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
  5. Add 1 μl T4-DNA Ligase
  6. Incubate 10-30 min at room temperature
  7. heat for 20 minutes at 80°C
  8. store at -20°C or directly proceed to transformation


Name of part Ratio Insert:Vector

= 3:1 or 1:1

Volume (μl)
X insert 1 S54 both 2 or 3
Y insert 2 PR 444 bzw. PR 668 2 or 3
Z vector Psb1A3 2 or 1
H2O 11

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation


Stored in Ligation-box, single parts stored in minipreps, verdaut-box

name of parts see above.

PCR

Investigators: Rüdiger

name templates primer
a pGex-6-P-1a P62+P63
b pGex-6-P-1b ´´
a10 pGex-G-P-1a ´´
b10 pGex-6-P-1b ´´

PCR did not work. New primer design.

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/18_August"