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

DNA Decoder

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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: 95 C: 5 mins. 95 C: 30 secs. 55 C: 30 secs. 72 C: 1 min. 72 C: 10 mins. 4 C: hold Numbers 2-4 were repeated 34 times.