Team:Freiburg/Notebook/1 August
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==<span style="color:grey;">Precipitator</span>== | ==<span style="color:grey;">Precipitator</span>== | ||
- | + | '''PCR''' | |
- | + | ||
- | { | + | {| style="border-spacing:0;" |
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Sophie | ||
+ | |||
+ | |||
+ | |||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 01.08.11 | ||
+ | |||
+ | |- | ||
+ | | colspan="2" style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: replay of PCR 19.07.11 (Date) 19.07.11 | ||
+ | |||
+ | (Name) Ruediger | ||
+ | |||
+ | |- | ||
+ | | colspan="2" style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: GFP Pbd | ||
+ | |||
+ | |} | ||
+ | PCR-Mixture for one Reaction: | ||
+ | |||
+ | For a 50 µl reaction use | ||
+ | |||
+ | |||
+ | {| style="border-spacing:0;" | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0 | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P28 | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P18 / P19/ P20 | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S 14 (GFP) | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end) | ||
+ | | style="border:0.0069in solid #000000;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| | ||
+ | |||
+ | |} | ||
+ | What program do you use? | ||
+ | |||
+ | Ruediger PCR (modified by Tobi and me. First 10 cycles with 55°C, next cycles with 62°C annealing temperature. | ||
+ | |||
+ | To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel. | ||
+ | |||
+ | |||
+ | How did you label the PCR-Product, where is it stored and what do you do next? |
Revision as of 11:29, 1 August 2011