DNA Amplification of specific target cDNA by PCR

DNA AMPLIFICATION OF SPECIFIC TARGET cDNA BY PCR

Last Update: December 2006

PROCEDURE

1. Into a 0.5 mL microcentrifuge tube placed on ice add: 1 mL RNA/cDNA hybrid or plasmid DNA (10-100 ng), 1 mL of sense primer 1 (50 pmoles/mL), and 1 mL of antisense primer 2 (50 pmoles/mL)

2. Then add to the tube the following 47 mL of pre-mix solution containing: 40.8 mL of dH₂O, 5 mL of 10X PCR buffer (0.1 M Tris-HCl, pH 8.3; 0.5M KCl; *15 mM MgCl₂), 1 mL of 10mM dNTP mix (pH-7), 0.2 mL of Taq DNA polymerase (1-5 U) (total volume: 50 mL)

*NOTE: The final concentration of MgCl2 in the reaction can vary from 0.5 to 5mM

3. Mix, spin, place in a thermocycler and immediately denature

4. PCR conditions: 94^oC for 5 minutes, followed by 20 to 30 cycles of PCR amplification: 94^oC for 50 secs, 44-58^oC for 50 seconds, and 72^oC for 90 seconds

5. End the reaction with a final incubation at 72^oC for 10 minutes

6. Keep at 4^oC until analyzed

7. Analyze 5 mL of the amplified sample using TBE/agarose gel electrophoresis