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

RNNA Decoder

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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.