Team:Freiburg/Notebook/7 September

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(Troubleshooting of the modified Lysis genes K124017)
 
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{{:Team:Freiburg/Templates/header}}
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<html>
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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/6_September">Previous entry</a>
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</div>
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<div id="notebook-title">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 7 September </a>
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<div id="notebook-next">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/8_September">Next entry</a>
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==Commons==
==Commons==
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*PR2+50b
*PR2+50b
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==<span style="color:green;">green light receptor</span>==
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===PCR of psB1C3===
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'''Investigators: Julia'''
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===NAME OF YOUR EXPERIMENT===
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[[File:Julias mit Dpn I verdaute Vektoren.jpg|350px|350px]]
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'''Investigators:NAME'''
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==<span style="color:green;">Green light receptor</span>==
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'''PCR'''
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{| style="border-spacing:0;"
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| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name: Julia
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&nbsp;
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| style="border:0.0139in solid #808080;padding:0.0194in;"| Date: 7.09.2011
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|-
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| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| Project Name: PcpcG
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|}
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&nbsp;
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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-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 32,5µl
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| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| H20
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| style="border:0.0139in solid #808080;padding:0.0194in;"|
 +
 +
|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 10µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 5x Phusion Buffer
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|
 +
 +
|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2.5µl
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Primer&nbsp;&nbsp;&nbsp;&nbsp; up
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC
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 +
|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2.5µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Primer&nbsp;&nbsp;&nbsp;&nbsp; dw
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC
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 +
|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 1µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| dNTPs
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 1µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| DNA-Template
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;Synechocystis genome
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 0.5 µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Phusion (add in the end)
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
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|}
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&nbsp;
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What program do you use?
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&nbsp;
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Temperature Time ( min)
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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;"| 94 C
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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;"| 5:00
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| style="border:0.0007in solid #000000;padding:0.0382in;"|
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|-
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 94 C
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 00:30
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| 30x
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|-
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 56 C
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 00:30
 +
| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"|
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|-
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 72 C
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 1:00
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| style="background-color:#ffffcc;border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"|
 +
 +
|-
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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;"| 72 C
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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;"| 7:00
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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;"|
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|}
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To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
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&nbsp;Size of expected gene : 238 bp
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
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PCR products were loaded onto a gel
PCR products were loaded onto a gel
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==<span style="color:red;">red light receptor</span>==
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[[File:Freiburg_07.08.11_Sophie.jpg]]
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===NAME OF YOUR EXPERIMENT===
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'''Investigators:NAME'''
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==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
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The concentration of '''''S4+S15 Nr1''''' should be about 1/2 of that of '''''M48''''', so I am going to use 2x and 6x the amount of M48 for the ligation.
The concentration of '''''S4+S15 Nr1''''' should be about 1/2 of that of '''''M48''''', so I am going to use 2x and 6x the amount of M48 for the ligation.
<br>
<br>
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There is no band to be seen for '''''S4+S15 Nr7''''', so the concentration is lower than 5ng/mikrol, so I am going to use all of it for the ligation (about 20-25mikrol) and change the reaction concentrations accordingly.
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There is no band to be seen for '''''S4+S15 Nr7''''', so the concentration is lower than 5ng/mikrol, so I am going to use all of it for the ligation (ca 25mikrol) and change the reaction concentrations accordingly.
<br>
<br>
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'''Procedure'''
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&nbsp;
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PCR tube:
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total volume 20 μl
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&nbsp;
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1. add H2O &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (17 μl -X-Y-Z)
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2. add 2 μl Ligase Buffer 10x
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3. add Insert&nbsp;&nbsp;&nbsp;
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4. add Vector &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (20ng needed)
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5. Add 1 μl T4-DNA Ligase
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6. Incubate&nbsp; 10-30 min at room temperature
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&nbsp;
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==

Latest revision as of 00:26, 22 September 2011


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