Team:Freiburg/Notebook/Methoden

From 2012.igem.org

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=<span style="color:#000000"> Methods =
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In this section we want to give you a chance to take a look at our protocols. From simple Agarose Gel over Colony PCR to transfection and SEAP measurment. Feel free to use the protocols for your own labwork and dont hesitate to contact us if you have any questions or problems regarding the protocols
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<div style=text-indent:40px>
<div style=text-indent:40px>
<a href="http://omnibus.uni-freiburg.de/~ds151/Transfection%20of%20HEK%20and%20SEAP%20measurement.pdf">Transfection and SEAP measurement.</a>
<a href="http://omnibus.uni-freiburg.de/~ds151/Transfection%20of%20HEK%20and%20SEAP%20measurement.pdf">Transfection and SEAP measurement.</a>
</html>
</html>
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==<span style="color:#000000"> First Extension PCR (Toolkit)==
 
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<p>
 
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This step is necessary to add the the necessary restriction sites to the Direpeats.
 
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<br/>
 
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Pipette the following volumes into a PCR- tube:
 
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{|cellpadding="5" cellspacing="0" border="1"
 
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|'''Component'''
 
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|'''Volume'''
 
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|-
 
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|Water, nuclease-free
 
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|15,75 µl
 
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|-
 
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|DMSO
 
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|0,75 µl
 
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|-
 
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|dNTPs
 
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|0,5 µl
 
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|-
 
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|Phusion Buffer
 
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|5,0 µl
 
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|-
 
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|Phusion Polymerase
 
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|0,25 µl
 
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|-
 
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|Primer forward
 
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|1,25 µl
 
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|-
 
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|Primer reverse
 
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|1,25 µl
 
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|-
 
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|Template
 
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|0,25 µl
 
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|-
 
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|'''Total'''
 
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|'''25 µl'''
 
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|}
 
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<br/>
 
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run the following PCR program:
 
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{|cellpadding="5" cellspacing="0" border="1"
 
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|'''step'''
 
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|'''temperatur'''
 
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|'''time'''
 
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|-
 
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|1.
 
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|98 °C
 
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|30 s
 
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|-
 
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|2.
 
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|98 °C
 
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|8 s
 
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|-
 
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|3.
 
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|68 °C
 
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|15 s
 
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|-
 
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|4.
 
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|72 °C
 
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|4 s
 
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|-
 
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|5.
 
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|98 °C
 
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|8 s
 
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|-
 
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|6.
 
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|70 °C
 
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|15 s
 
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|-
 
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|7.
 
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|72 °C
 
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|5 s
 
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|-
 
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|8.
 
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|72 °C
 
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|5 min
 
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|-
 
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|9.
 
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|4 °C
 
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|store
 
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|-
 
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|}
 
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Repeat step 2. - 4. 15 times ans step 5. - 7. 30 times.
 
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<br/>
 
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</p><br>
 
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==<span style="color:#000000">Second Extension PCR (Toolkit)==
 
-
<p>
 
-
This step is necessary to add enzyme- binding –sites to the outer restriction sites.
 
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Use the product of the first extension- PCR as template.
 
-
<br/>
 
-
Pipette the following volumes into a PCR- tube:
 
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</p><br>
 
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==<span style="color:#000000">PCR Purification==
 
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<p>
 
-
 
-
bla
 
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</p><br>
 
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==<span style="color:#000000">Gel Run==
 
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<p>
 
-
 
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bla
 
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</p><br>
 
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==<span style="color:#000000">Ligation==
 
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<p>
 
-
 
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bla
 
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</p><br>
 
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==<span style="color:#000000">Colony PCR==
 
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<p>
 
-
 
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bla
 
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</p><br>
 
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==<span style="color:#000000">Mini-Prep==
 
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<p>
 
-
 
-
bla
 
-
 
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</p><br>
 

Revision as of 22:12, 25 September 2012





Methods

In this section we want to give you a chance to take a look at our protocols. From simple Agarose Gel over Colony PCR to transfection and SEAP measurment. Feel free to use the protocols for your own labwork and dont hesitate to contact us if you have any questions or problems regarding the protocols

Transfection and SEAP measurement.

Retrieved from "http://2012.igem.org/Team:Freiburg/Notebook/Methoden"