Nested PCR Amplification

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

Followed by:

Final extension 72°C for 7 min

Indefinite hold 4°C, until samples are removed

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