The second PCR amplification step uses the nested GSP2 Anchor Primer to ensure the specific amplification of the correct 3’ L1H-specific fragments and add an additional anchor sequence to the ends of the amplified DNA.
- Dilute the reaction from the First Round of PCR (Step 4 above) 10-fold with water. Use 2 µl of the diluted First Round PCR reaction for the Second Round of Nested PCR.
- Prepare the Second Nested PCR Reaction Mix as follows on ice in a 50 μl thin wall PCR tube:
Component Volume 10-fold diluted DNA from 1st PCR (Step 4) 2 µl HF DNA Polymerase Buffer (5X) 10 µl dNTP Mix (50X) 1 µl GSP2 Anchor 1 µl Adapter Primer 1 µl PCR-Grade Water 34.5 µl HF DNA Polymerase (100X) 0.5 µl Total 50 µl
- Mix gently and centrifuge briefly to collect droplets.
- Amplify the Second Nested PCR Reaction Mix 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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