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.

  1. 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.
  1. 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
  1. Mix gently and centrifuge briefly to collect droplets.
  1. 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
Last modified: Mar 08, 2017

Need more help with this?
Contact Us

Thanks for your feedback.