1 2 3 4 5 6

Tube

cDNA template 0(neg) 5 ul(pos)

dH2O 5 ul 0 ul

Rxn mix 45 ul 45 ul

3. Add one drop of paraffin oil to each tube to prevent evaporation. 4. Run in PCR machine (anneal at60ºC for GAPDH).

Program Stage1 Stage 2

94ºC 94ºC 60 ºC 72ºC 72ºC 35ºC

2 min. 30 sec. 1 min. 1 min. 5 min. 30 min.

1 cycle 30 cycles

Stage 3 Stage 4

1 cycle 1 cycle

5. Store at -20º C. 6. Run 10 ul sample + 4 ul loading dye on 1.5% agarose minigel. 4 ul PCR markers + 4 ul dH20 + 4 ul loading dye