IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/19

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July 19, 2010
We redid the PCR's from Saturday. The primers were not diluted to 1/10 their original concentrations.
 * 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:
 * 1) 95 C: 5 min.
 * 2) 95 C: 30 sec.
 * 3) 51 C: 30 sec.
 * 4) 72 C: 1.5 min.
 * 5) 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