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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

{| width="800"
 * style="background-color: #cdde95;" align="center"|
 * style="background-color: #cdde95;" align="center"|




 * align="center" style="background-color: #e5edc8;" |

title=Search this Project


 * colspan="2" style="background-color: #F2F2F2;" align="right"|Customize your entry pages 
 * colspan="2"|
 * colspan="2"|
 * colspan="2"|

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.


 * }