User:Nelson Augusto Berrocal/Notebook/WiFi Coli 2010 Wet Lab/2010/06/08

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June 08th, 2010

I made a PCR following the protocol of the last day with a few changes: I add 3μL of template DNA instead 4μL (because of the lacking of template DNA), and I change the time for each cycle.

95°C-4:30min------- | ---95°C-45seg-------60°C-45seg--------72°C-1:45seg--- (30 cycles)| 72°C-5:00min -----------4°C---->
Soon after finishing my PCR I ran an agarose gel to .8% to prove that the biobrick BBa_K098010 was amplified; the gel lanes were as following:
Ladder | Control + | Control - | Biobrick Amplified | Ladder
I used 1μL of ladder, and the other lanes were filled with 5μL of DNA (each of them, control as well as biobrick) and 1.5μL loading buffer.
RESULTS
We have obtained at least a 1.5kb band corresponding to biobrick BBa_K098010, now I'm gonna make a High Fidelity PCR in order to obtain the functional biobrick.



I made a High Fidelity PCR with the plasmid pSB4C5 in order to amplify the biobrick BBa_K098010; I followed the next protocol:
I prepared two solution; the first one was prepared as following:
3μL template DNA
6μL Buffer 3.3x
3μL Mg(Ac)2
2.5μL dNTPs
2.5μL Suffix
2.5μL Preffix
H2O
A total of 30μL

The second solution was made as follows:
10.5μL H2O
9μL Buffer 3.3x
0.5μL Rtth
Total: 20μL

First I placed the first solution in the thermomixer and after 5 minuts at 95°C, I placed the second solution to begin the reaction; at cycle 14 the thermomixer suddenly turned off so I restarted the cycles (only 16 cycles), to accomplish the 30 cycles, I hope everything is OK.
Once the PCR was done, I made a gel to prove it was OK:
Ladder 1μL | Positive Control | Biobrick | Ladder 1μL.
RESULTS
FAILED


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