Team:Freiburg/Notebook/15 July

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===<span style="color:orange;">Lysis cassette</span>===
===<span style="color:orange;">Lysis cassette</span>===
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Digestion of Quickchange
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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: 15.07.2011
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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;"| Continue from Experiment 14.07.2011
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Quickchange PCR
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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: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device
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|}
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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;"| 4,5μl
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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>O
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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;"| 4μl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Buffer, NEB4
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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;"| 4μl
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x)
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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;"| 1,5 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;"| Enzym 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;"| DpnI
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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;"| 21 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;"| DNA
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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;"| Quickchange PCR
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|}
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35 μl total volume
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Incubate for about 1h at 37°C + Heat inactivation at 80°C for 20min.
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<br>
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===Quick change===
===Quick change===

Revision as of 14:59, 18 August 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

Meeting

attendants: Tobi, Theo, Julia, Rüdiger, Jakob, Sandra

blue light receptor

already done:

  1. Transformation of Lov-Tap in cells.


To-do:

  1. Cloning of promotor (medium) in Lov-Tap part and perform a transformation. Plating out of the cells on trypthophan-free medium.



Lysis cassette

Digestion of Quickchange


Name: Theo


Date: 15.07.2011
Continue from Experiment 14.07.2011

Quickchange PCR

Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device
4,5μl H2O
4μl Buffer, NEB4
4μl BSA (10x)
1,5 μl Enzym 1 DpnI
21 μl DNA Quickchange PCR

35 μl total volume


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


Quick change

Investigators: Theo

already done:

  1. repressor part (BBa_K098995) has no bases between RBS (B0034) and start codon (K098997), resulting in no translation.
    • quickchange of the repressor part to insert 6bp between RBS and start codon

To-do:

  1. DpnI digestion to digest the DNA template (methylated DNA) of the PCR to have only the new synthesized DNA strand.
  2. After digestion with DpnI, transformation of cells with the corrected part (BBa_K08995)



Precipitator

PCR


Name:

Ruediger

Date:

18.07.2011

Continue from Experiment (Date)PCR 1507

(Name) Ruediger

Project Name:

GFP Pbd

PCR-Mixture for one Reaction:

For a 50 µl reaction use


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

What program do you use?

10x (95C-41/52C-72C) + 25x ((95C-60C-72C)


How did you label the PCR-Product, where is it stored and what do you do next?


Reactions:

lane 1

quick load braod range marker

lane 2

empty

lane 3

P28+P18+M14.1

lane 4

P28+P19+M14.1

lane 5

P28+P20+M14.1


Results:

did not work well.

Strange band size in lane 3. 4 and 5 did not work.

Freiburg11 7 16 2011 2 52 42 PM.Jpg Freiburg11 Phusion ladder.JPG

                       

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