IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/17

RNA Decoder

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July 17, 2010 Francis couldn't wait until Monday to get things rolling and came in on Saturday to get some work done. He did the following PCRs (and diluted the primers to 1/10 concentration): RXN 1 (MicA) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 2 (OmpA) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 3 (MicF): 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 4 (OmpF) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 5 (GadY) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 6 (GadX) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 7(Hfq) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 32.8 uL dH2O Total= 50 uL RXN 8 (EGFP) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 1 uL PXG-10 Template 41.8 uL dH2O Total= 50 uL RXN 9 (Polymerase Control) 10 uL 10X Pfu Buffer 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 10 uL MG1655 Template 33.2 uL dH2O Total= 50 uL RXN 10 (Template Control) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 0.4 uL Primer Forward 0.4 uL Primer Reverse 1.0 uL 4X dNTP 42.8 uL dH2O Total= 50 uL RXN 11 (Primer Control) 10 uL 10X Pfu Buffer 0.4 uL Pfr DNA Polymerase 1.0 uL 4X dNTP 10 uL MG1655 Template 33.6 uL dH2O Total= 50 uL The PCR setting that was used: 95 C: 5 min. 95 C: 30 sec. 51 C: 30 sec. 72 C: 1.5 min. 72 C: 10 min. 4 C: hold Numbers 2-4 were repeated 32 times. The Concentrations of the templates were: PXG-10: 475 ng/uL MG1655: 2.8 ng/uL The Concentrations of the cloned genes were: Hfq: 44.70 ng/uL OmpA: 1.66 ng/uL GadY: 15.88 ng/uL OmpF: 2.65 ng/uL EGFP: -1.47 ng/uL MicF: 16.78 ng/uL MicA: 7.60 ng/uL GadX: 43.69 ng/uL