Reagents for one 8uL Rxn:
10X Advantage Buffer 0.63uL 10 mM dNTPS 0.135uL 20uM primer1 0.10125uL 20uM primer2 0.10125uL Advantage Taq (5U/uL) 0.045uL ddH2O 3.9875uL Template 3.0uL from 3:150 dilution in H2O from Original bacterial cDNA frozen stock plate Use the following primer pairs: 0T2a vector: PM001a 5' GTCGACGTTAGAACGCGGCTAC 3' PM002a 5' GGGTTAAATTCCCGGGTACTGC 3' BS vector: SK-30 5' GGGTAACGCCAGGGTTTTCC 3' SKMet 5' ATGACCATGATTACGCCAAGC 3'
Advantage Buffer and cDNA Polymerase Mix (#8417-1) are available from Clontech Laboratories, Inc. We have also successfully used DyNAzyme EXT Polymerase (F-505L) and EXT Buffer with Mg2+ (F-514L) available from Finnzymes and MJ Research, Inc. If DyNAzyme is used, please change the extension temperatures in the PCR profile to 72 C instead of 68 C.
95 C 1 min 95 C 0.5min 68 to 58 C 0.75min Touchdown for 5 cycles 68 C 3 min 95 C 1 min 58 C 0.75min Auto extend for 28 cycles 68 C 4 min + 0.1min 68 C 10 min 4 C soak