A single PCR with 35 cycles will not often generate enough specific product to be detectable by ethidium bromide staining. A second nested PCR is strongly recommended in order to enhance the product yield and the specificity of the amplification.
1. Dilute a 5-pL aliquot of the primary PCR into 495 pL of TE buffer.
2. Equilibrate the thermal cycler to 94°C.
3. Label with appropriate nomenclature the thin-wall PCR tubes sitting on ice.
4. Add the following to a 1.5-mL Eppendorf tube sitting on ice (reaction mix, composition/sample):
Sterilized, deionized water 31.5 pL
10X PCR buffer 5 pL
25 mM MgCl2 3 pL
10 mM dNTP mix 1 pL
10 mM ALK3 primer (see Note 10) 2 pL
10 mM AAP primer 2 pL
5. Add 0.5 pL of Taq polymerase/sample and gently mix all the components by up and down pipetting.
6. Dispense 45 pL of the reaction mix in each of the corresponding PCR tubes.
7. Add 5 pL of the dC-tailed cDNA to the corresponding tubes except for the negative control where 5 pL of sterilized water must be added.
8. Transfer the tubes directly from the ice to the pre-equilibrated thermocycler at 94°C.
9. Perform 35 cycles of PCR:
Pre-amplification denaturation 94°C for 2 min Cycle:
Denaturation 94°C for 1 min
Annealing 64°C for 1 min
Extension 72°C for 1 min, 30 s
Final extension 72°C for 7 min
Indefinite hold 4°C, until samples are removed
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