The First Round of PCR selectively amplifies DNA fragments that have the T-Adapter on one end and the 3’ L1-specific sequences on the other. These fragments are amplified by PCR using the HF (High Fidelity) Polymerase for 15 cycles in 50 µl using the GSP1 primer and T-Adapter primer.
- Use half (15 µl) of the T-Adapter-ligated 3’ L1H DNA sample from Step 3. Save the other half in case you may need to repeat the experiment due to potential problems during the next steps of the protocol. Prepare the following reaction mix in a 50 μl thin-wall PCR tube:
Component Volume T-Adapter-ligated DNA from Step 3 15 µl HF DNA Polymerase Buffer (5X) 10 µl dNTP Mix (50X) 1 µl GSP1 Primer 1 µl Adapter Primer 1 µl PCR-Grade Water 21.5 µl HF DNA Polymerase (100X) 0.5 µl Total 50 µl
- Mix gently and centrifuge briefly to collect droplets.
- Amplify adapter-ligated 3’ L1H DNA products using the following PCR program:
98°C, 60 seconds 1 cycle 98°C, 15 seconds
62°C, 15 seconds
72°C, 30 seconds
15 cycles 72°C, 60 seconds 1 cycle 4°C, ∞ 1 cycle
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