IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/20

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July 20, 2010
Today we retried the EGFP PCR because we were not able to clone it out yet. We did two of the same reactions for the EGFP:


 * RXN 1 EGFP (Done twice in two separate tubes)
 * 0.5 uL Template (pXG-10 475 ng/uL)
 * 5.0 uL 10X pfu Buffer
 * 0.4 uL Primer 52820847 (1/10 dilution)
 * 0.4 uL Primer 52820848 (1/10 dilution)
 * 1.0 uL 4X dNTP
 * 0.2 uL pfu DNA polymerase (Turbo)
 * 42.5 uL dH2O
 * Total: 50 uL


 * RXN 3 Control 1 (no template)
 * 5.0 uL 10X pfu Buffer
 * 0.4 uL Primer 52820847 (1/10 dilution)
 * 0.4 uL Primer 52820848 (1/10 dilution)
 * 1.0 uL 4X dNTP
 * 0.2 uL pfu DNA polymerase (Turbo)
 * 43.0o uL dH2O
 * Total: 50 uL


 * RXN 4 Control 2 (no primers)
 * 0.5 uL Template (pXG-10 475 ng/uL)
 * 5.0 uL 10X pfu Buffer)
 * 1.0 uL 4X dNTP
 * 0.2 uL pfu DNA polymerase (Turbo)
 * 43.3 uL dH2O
 * Total: 50 uL


 * RXN 5 Control 3 (No polymerase)
 * 0.5 uL Template (pXG-10 475 ng/uL)
 * 5.0 uL 10X pfu Buffer
 * 0.4 uL Primer 52820847 (1/10 dilution)
 * 0.4 uL Primer 52820848 (1/10 dilution)
 * 1.0 uL 4X dNTP
 * 42.7 uL dH2O
 * Total: 50 uL

The PCR machines settings were:
 * 1) 95 C: 5 mins.
 * 2) 95 C: 30 secs.
 * 3) 55 C: 30 secs.
 * 4) 72 C: 1 min.
 * 5) 72 C: 10 mins.
 * 4 C: hold

Numbers 2-4 were repeated 34 times.