Team:Freiburg/Notebook/5 September

From 2011.igem.org

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==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
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'''Digestion'''
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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:Rüdiger
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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: 05.09.
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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 Digestion Date 31.08. Name Rüdiger
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Experiment
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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: Precipitator
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|}
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Procedure
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# add H<sub>2</sub>O (38μl-DNA )
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# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
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# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
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# DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
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# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
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# heat for 1-2 hours 37°C (6 hours if time)
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# heat for 20 minutes 80°C (inactivation of enzymes)
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# keep at 4°C if you cannot continue
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Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
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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;"| Sample Name
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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 concentration (μg/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;"|
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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;"|
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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;"|
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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;"|
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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;"|
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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;"|
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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;"|
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|}
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Restriction enzymes you need to cut the vector, insert1 and insert 2:
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{| style="border-spacing:0;"
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| 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
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| 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;"| Vector (+Antarctic phosphatase 1 μl +AP buffer 5,6 μl)
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| 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;"| Insert
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(+Dpn1 1 μl)
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| 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;"| Enzyme1
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| style="border:0.0007in solid #000000;padding:0.0382in;"| Enzyme2
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 1
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PGEX David
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 30
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Bam H1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 2
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PGEX David
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 30
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Bam H1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 3
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PGEX David
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 30
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Bam H1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 4
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| C3
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 8
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Spc1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 5
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PET DUET
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 12
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Pst1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 6
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PET DUET
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 22
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Pst1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 7
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| PET DUET
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 20
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Pst1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 8
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| C3
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 20
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Spc1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 9
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| C3
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 20
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Spc1
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 10
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| C3
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 15
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| EcoR1
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| 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;"| Spc1
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|}
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'''Documentation:'''
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Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.
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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;"| Repeated digeston from 31.08..
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|}
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Run a gel to verify if the part is cut out.
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===Miniprep===
===Miniprep===

Revision as of 20:36, 21 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

transformation with reporter parts

Investigators:Julia

transformation with iGEM parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15016 BBa_I15016] which is B0032.ECFP
and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15017 BBa_I15017 ]which is B0032.EYFP.
Tomorrow the green light promotor from PCR of 29_August should be inserted infront of the reporter proteins CFP and YFP.

blue light receptor

Miniprep

Investigators:Sophie
continue from: picking clones; 04.09.11

Testdigest showed no inserts

Ligation

Investigators: Sophie
again ligation as last time (02.08.11)

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo


Minipreps of the first inoculations were applied on a 1% agarose gel without digesting them to see if they have an insert of the modified Lysis genes K124017. Nr1, 5 and 7 were sent for sequencing since the size seems to be about right.

  • Note: S4 is the B0034 RBS (ribosome binding site), S15 is the non-modified K124017 Lysis Cassette without RBS


Freiburg11 9 7 2011 Gel von S4plusS15 ohne Verdau.Jpg


Precipitator

Digestion


Name:Rüdiger Date: 05.09.
Continue from Digestion Date 31.08. Name Rüdiger

Experiment

Project Name: Precipitator

Procedure


  1. add H2O (38μl-DNA )
  2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
  3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
  4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
  5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
  6. heat for 1-2 hours 37°C (6 hours if time)
  7. heat for 20 minutes 80°C (inactivation of enzymes)
  8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:


Sample Name DNA concentration (μg/μl)

Restriction enzymes you need to cut the vector, insert1 and insert 2:


Name Vector (+Antarctic phosphatase 1 μl +AP buffer 5,6 μl) Insert

(+Dpn1 1 μl)

Enzyme1 Enzyme2
1 PGEX David 30 EcoR1 Bam H1
2 PGEX David 30 EcoR1 Bam H1
3 PGEX David 30 EcoR1 Bam H1
4 C3 8 EcoR1 Spc1
5 PET DUET 12 EcoR1 Pst1
6 PET DUET 22


EcoR1 Pst1
7 PET DUET 20 EcoR1 Pst1
8 C3 20 EcoR1 Spc1
9 C3 20 EcoR1 Spc1
10 C3 15 EcoR1 Spc1

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


Repeated digeston from 31.08..

Run a gel to verify if the part is cut out.


Miniprep

Investigators: Sophie
continue from experiment: Transformation; 03.09.11
testdigest showed no inserts

Trafo

Investigators. Sandra

Repeat of the trafo of:

  1. pGEX vector (Amp. resistance)
  2. pGEX vector (Amp. resistance)
  3. pGEX vector (Amp. resistance)
  4. pSB1C3
  5. pET Duet vector (Amp. resistance)
  6. pET Duet vector (Amp. resistance)
  7. pET Duet vector (Amp. resistance)
  8. pSB1C3
  9. pSB1C3
  10. pSB1C3

                       

Trafo

Investigators. Sandra

Repeat of the trafo of:

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/5_September"