From each of the three plates of growth two colonies (one white and one red) were grown over night in YPAD at 30*. The cells were then spun down and the broth was removed from the cells. Buffer P1 (250 micro liters) and glass beads were mixed with the cells and shook for ten minutes. Buffer P2 (250 micoL) was added to the tube and mixed. Then after 4 minutes buffer N3 (350 micoL) was added and mixed. The tube was then centrifuge at 13,000 rpm for 10 minutes. The top liquid was then removed and place in a spin column and spun for a minute (at 13,000 rpm). The DNA was then washed with PB (500 microL) and centrifuged for a minute. Buffer PE (750 mL)was added and centrifuged for a minute. The column was then spun dry for a minute. Buffer EB(30 microL) was added and sat for five minutes. The DNA was then spun down into a clean tube.
Before we could send our plasmids in to be coded we had to amplify the DNA. Taq master mix (9 microL), plasmid DNA from above (1 microL), Primer mix Seq (1 microL), and water (7 microL) were mixed and PCRed with the following program:
95* for 2 minutes
(95* for 30 seconds, 54* for 30 seconds, 72* for 1 minute) 35 times
72* for 10 minutes
The ender PCR was then ran on a gel check and then purified.