IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/07/13

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dspB Track Marianne and Vicki PCR II Dilute PCR product, PCR again, and gel verify Dilution for each sample: 2uL of PCR product + 10uL of H2O = 12uL (1 in 5 dilution) Protocol: common protocols for PCR Table 1. Microcentrifuge Tubes 1 2 3 4 5 6 7 8 9 10 11 12 MMA: 6 diluted samples + 1 extra + 1 water control = 8 samples MMB: 6 diluted samples + 1 extra = 7 samples Table 2. PCR Tubes Label Content A 1 B 2 C 3 D 4 E 5 F 6 G 7 H 8 I 9 J 10 K 11 L 12 M 13 Table 3. Master Mix A Reagent 1X rxn volume (uL) Master Mix 10x rxn buffer 2.5 x8 20 10uM FW primer 2 x8 16 10uM RE primer 2 x8 16 10mM dNTP 3 x8 24 sdH2O 10.3 x8 92.4 Taq polymerase 0.2 x8 1.6 DNA 5 x8 40 Total 25 200 Table 4. Master Mix B Reagent 1X rxn volume (uL) Master Mix 10x rxn buffer 2.5 x7 17.5 10uM FW primer 2 x7 14 10uM RE primer 2 x7 14 10mM dNTP 3 x7 21 sdH2O 10.3 x7 72.1 Taq polymerase 0.2 x7 1.4 DNA 5 x7 35 Total 25 175 PCR Cycles: 95C @ 2 min Cycle 30x: 95C @ 30 sec 65C @ 10 sec 72C @ 80 sec 72C @ 10 min 10C @ hold Start: 1225 End: 1355 Gel verification Protocol: in iGEM training manual Changes: Made 1% agarose gel instead of 0.8% gel Gel orientation: Gel orientation A B C D E F 100bp ladder G H I J K L M Machine conditions: 110V, 45 min, 0.5X TBE Buffer Results: [[Image:|300px]] Vicki Ma 20:37, 13 July 2010 (EDT)