Team:Freiburg/Notebook/23 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook/22_July">Previous entry</a>
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<div id="notebook-title">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 23 July </a>
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/25_July">Next entry</a>
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==<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: 23.07.2011
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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 22.07.2011
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Quickchange V.3 with correct Lysis Template
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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 V.3 with correct Lysis Template
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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;"| 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;"| 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;"| 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;"| 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;"| 2,5 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;"| 37,5 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;"| 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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50 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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===Transformation''' '''===
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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: 23.07.2011
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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 23.07.2011 Digestion of Quickchange V.3
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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: Quickchange V.3 with correct lysis template
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|}
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Procedure
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# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
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# thaw cells on ice 20 minutes
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# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
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# Incubate for 30 minutes on ice
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# Heat at 42°C for 60 sec
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# Incubate on ice for 5 minutes
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# Add 200 μl LB Broth
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# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
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# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
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'''Documentation:'''
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Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance 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;"| It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.
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This time the correct DNA template was used, ie S11 (K098995). The backbone vector is psB1A3 (Amp resistance)
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|}
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<br>
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<br/>
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 01:00, 22 September 2011


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


Lysis cassette

Digestion of Quickchange

Name: Theo


Date: 23.07.2011
Continue from Experiment 22.07.2011

Quickchange V.3 with correct Lysis Template

Project Name: Quickchange V.3 with correct Lysis Template
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 1h at 37°C + Heat inactivation at 80°C for 20min.


Transformation

Name: Theo Date: 23.07.2011
Continue from 23.07.2011 Digestion of Quickchange V.3

Experiment

Project Name: Quickchange V.3 with correct lysis template

Procedure


  1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
  2. thaw cells on ice 20 minutes
  3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
  4. Incubate for 30 minutes on ice
  5. Heat at 42°C for 60 sec
  6. Incubate on ice for 5 minutes
  7. Add 200 μl LB Broth
  8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
  9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.


It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.


This time the correct DNA template was used, ie S11 (K098995). The backbone vector is psB1A3 (Amp resistance)



                       

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