Team:Freiburg/Notebook/3 August

From 2011.igem.org

(Difference between revisions)
(→Quickchange of BBa_124017 to insert RBS (B0034) and 6bp in front of the first ATG)
(→Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG)
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'''Digestion of Quickchange'''
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===='''Quickchange 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: Theo
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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: 03.08.2011
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|-
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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 of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG
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|}
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PCR-Mixture for one Reaction:
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For a 50 µl reaction use
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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;"| 32,5µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 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;"| Name
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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;"| 10µl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6x Phusion HF Buffer
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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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| 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;"| P55 (RBS2LysV2up) (1:10)
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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;"| 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 dw
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P56 (RBS2LysV2dw) (1:10)
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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;"| 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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|-
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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;"| S49 (Composite part -modified K098995+K124017) (->diluted to 5ng/µl)
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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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What program do you use?
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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;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>95°C - 15 sec</center>
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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;"|
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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;"| <center>95°C - 5 min</center>
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>55°C / 66°C - 15 sec</center>
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°C -5 min</center>
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>4°C - ∞</center>
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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;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°C - 1’ + 2’’</center>
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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;"|
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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;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''20x'''</center>
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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;"|
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|}
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<br>
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<br/>
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{{:Team:Freiburg/Templates/footer}}
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===Digestion of Quickchange===

Revision as of 15:20, 9 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


Testdigest

Investigators:Jakob, Julia

Name:

Jakob

Date:

02.08.2011

Continue from Experiment 02.08.2011

Miniprep

Project Name: green light receptor, testdigest of CcaR with BamHI

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 1 BamHI
0,5 μl Enzym 2 -
3 μl DNA 3

10 μl total volume


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

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!


Freiburg 11 8 3 2011 7 09 46 PM.jpg200px


CcaR digested with BamH1 are negativ

  • To-do: quickchange (scroll down)


Quickchange


Investigator:Jakob

PCR


Name:

Jakob

Date:

03.08.2011

Continue from Experiment:
Project Name:

Quickchange CcaR

PCR-Mixture for one Reaction:

For a 50 µl reaction use


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



results of transformation from 2nd Aug

Investigators:Julia
transformation worked!

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



red light receptor

Colony picking

Investigator:Jakob

Picked some colonies for grow over night

  • to-do: Miniprep

Lysis cassette

Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG


Primers for Quickchange

  • Forward Primer: GGCGGTGATAATGGTTGCTAaagaggagaaaCTAGAGATGCCAGAAAAAACATGAC
  • Reverse Primer: GTCATGTTTTTTCTGGCATCTCTAGtttctcctcttTAGCAACCATTATCACCGCC


Quickchange PCR

Name: Theo


Date: 03.08.2011
Project Name: Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 6x Phusion HF Buffer
2.5µl Primer fw P55 (RBS2LysV2up) (1:10)
2.5µl Primer dw P56 (RBS2LysV2dw) (1:10)
1µl dNTPs
1µl DNA-Template S49 (Composite part -modified K098995+K124017) (->diluted to 5ng/µl)
0.5 µl Phusion (add in the end)

What program do you use?


95°C - 15 sec
95°C - 5 min
55°C / 66°C - 15 sec
72°C -5 min
4°C - ∞
72°C - 1’ + 2’’
20x



                       

Digestion of Quickchange

Name: Theo


Date: 03.08.2011
Continue from Experiment 03.08.2011

Ready Lysis Cassette (Part S49) + RBS

Project Name: Ready Lysis Cassette (Part K124017) + RBS (B0034)
5μl Buffer, NEB4
5μl BSA (10x)
2,5 μl Enzym 1 DpnI
37,5 μl DNA Quickchange PCR

50 μl total volume


Incubate for about 1,5h at 37°C + Heat inactivation at 80°C for 20min.

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME

                       

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