McClean: Takara PCR

January 2010 1 of 1 McClean Lab Protocol Takara PCR Reagents 1. 5X Takara Primestar Buffer 2. DNTPS (2.5mM each) 3. Primer 1 (10μM) 4. Primer 2 (10μM) 5. Plasmid DNA (20ng/μl concentration) 6. Takara Primestar Polymerase 7. Sterile Water For each 50ul reaction: Reagent Vol. # Reactions Total Reagent Vol. 1. 5X Takara Primestar Buffer 10μl x = 2. DNTPS (2.5mM each) 4μl x = 3. Primer 1 (10μM) 1μl x = 4. Primer 2 (10μM) 1μl x = 5. Plasmid DNA (20ng/μl concentration) 1μl x = 6. Takara Primestar Polymerase 0.5μl x = 7. Sterile Water 32.5μl x = 50μl Reaction Vol. Total Vol. Run PCR Program (labeled takarapcr on thermocycler) 94.0°C for 2 min 98.0°C for 10 sec 53.0°C for 15sec Repeat 30 times 72.0°C for 2min 30sec 72.0°C for 1min 4°C forever