User:Nelson Augusto Berrocal/Notebook/WiFi Coli 2010 Wet Lab/2010/06/09

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June 09th, 2010

I made a High Fidelity PCR from biobrick BBa_K098010 (which contains PcyA and Ho1) in order to obtain a useful biobrick; I followed the next protocol:
I made two reactions:
3μL template DNA
6μL Buffer 3.3x
3μL Mg(Ac)2
2.5μL dNTPs
2.5μL Suffix
2.5μL Preffix
10.5μL H2O
Total 30μL
The last reaction is placed in the thermomixer during 5 min to 95°C (Hot-start) and then the second reaction is added, the second tube contains the enzime Rtth.
9μL Buffer 3.3x
0.5μL Rtth Polymerase
10.5μL H2O
The PCR timing was adjusted like this:
95°C-5min----- | 95°C-45sec---------60°C-45sec---------72°C-1.45min---- | -------72°C-5min-------4°C--->
Once the PCR ended, I made an agarose gel to 0.8%:
Ladder - Positive Control - Biobrick - Ladder
But finally the gel didn´t show the ampliffied biobrick although the Positive Control was OK.



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