IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/12

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8/12 Ethanol Got plasmid PACYC Duet-pdc-adhb in strain DH10b and strain BL21 from Nielsen's lab to use for ethanol production Plated both in preparation to transform plasmid into BL21 Strain. Transformed ligation mixture Ligation mixture - Control + Control (ACC Plasmid) PCR Amplification of ACC parts First, the dry primers were made into 100 μM master stocks. 1 μL was added per .1 nm. For example, a 31.2 nm sample had 312 μL added. This master stock was then diluted into working stocks of 5 μM. This was done by adding 1 μL of master mix into 19 μL H2O. This was done for all eight primers , four forward and four reverse. The ACC plasmid was then diluted from 54.2 μg/μL to about 1 μg/μL through a 1/50 dilution. PCR mix for all four reactions (accA, accB, accC, and accD) PCR reaction Reagent 2x GoTaq Master Mix 12.5 Forward Primer (working stock) 1.0 Reverse Primer (working stock) 1.0 Template DNA (Diluted) 1.0 H2O 9.5 Total 25.0 Initial denature step of 95 °C for 2 min PCR cycle: 95 °C for 30 sec -> 45 °C for 45 sec -> 73 °C for 80 sec Final annealing step at 73 °C for 5 min