IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/01

{| width="800"
 * style="background-color: #EEE"|[[Image:Illinois09Decoder.jpg|250px]] RNNA Decoder
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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

July 1, 2010
We ran another PCR for MicA, MicF, OmpA, OmpF, and Hfq. We also ran two controls for the experiment: one without polymerase and one without template. Their uL amounts were replaced with dH2O. The procedure we followed:


 * 10X Buffer 5.0 uL
 * Pfu Turbo DNA Polymerase 0.4 uL
 * Primer Forward 0.5 uL
 * Primer Reverse 0.5 uL
 * dNTP 1.0 uL
 * Template (MG1655) 0.5 uL
 * dH2O 42.1 uL
 * Total= 50 uL

A master mix instead of adding each of those amounts to each tube. The master mix was made as such (MM for 5 RXNs):
 * 10X Buffer 25uL
 * Pfu Turbo DNA Polymerase 2 uL
 * dNTP 5 uL
 * Template (MG1655) 2.5 uL
 * dH2O 210.5 uL

We then ran the PCR at these settings:
 * 95 C: 5 mins.
 * 95 C: 30 sec.
 * 58 C: 30 sec.
 * 72 C: 1 min.
 * 4 C: hold


 * Steps 2-4 were repeated 32 cycles.

Once the PCR was complete we ran a gel for the trials.