IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/06/30

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June 30, 2010
Today we tried cloning out MicA, MicF, OmpA, OmpF, and Hfq. The procedure we followed was:
 * 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 also ran controsl for each reaction: one without template, and one without polymerase. In each water replaced the missing material.

The PCR settings were:
 * 95 C= 5 min.
 * 95 C= 30 secs.
 * 55 C= 30 secs.
 * 72 C= 1 min.
 * 72 C= 5 min.
 * 4 C= Hold


 * Steps 2-4 repeated 30 cycles

We then ran a electrophoresis gel of my PCR results.