Team:Freiburg/Notebook/Methoden

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(First Extension PCR (Toolkit))
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<a href="http://omnibus.uni-freiburg.de/~ds151/Transfection%20of%20HEK%20and%20SEAP%20measurement.pdf">Transfection and SEAP measurement.</a>
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==<span style="color:#000000"> First Extension PCR (Toolkit)==
==<span style="color:#000000"> First Extension PCR (Toolkit)==

Revision as of 18:43, 25 September 2012




Transfection and SEAP measurement.

First Extension PCR (Toolkit)

This step is necessary to add the the necessary restriction sites to the Direpeats.
Pipette the following volumes into a PCR- tube:

Component Volume
Water, nuclease-free 15,75 µl
DMSO 0,75 µl
dNTPs 0,5 µl
Phusion Buffer 5,0 µl
Phusion Polymerase 0,25 µl
Primer forward 1,25 µl
Primer reverse 1,25 µl
Template 0,25 µl
Total 25 µl


run the following PCR program:

step temperatur time
1. 98 °C 30 s
2. 98 °C 8 s
3. 68 °C 15 s
4. 72 °C 4 s
5. 98 °C 8 s
6. 70 °C 15 s
7. 72 °C 5 s
8. 72 °C 5 min
9. 4 °C store

Repeat step 2. - 4. 15 times ans step 5. - 7. 30 times.



Second Extension PCR (Toolkit)

This step is necessary to add enzyme- binding –sites to the outer restriction sites. Use the product of the first extension- PCR as template.
Pipette the following volumes into a PCR- tube:


PCR Purification

bla


Gel Run

bla


Ligation

bla


Colony PCR

bla




Mini-Prep

bla