This step primes and elongates the 3’ regions of L1 elements (3’ L1) using the GSP1 primer which sits ~100 bp from the start of the L1 element (Figure 2A). The Taq DNA Polymerase needs to be used in this reaction since it adds a 3’-adenine overhang to the ends of the extended double-stranded fragments. Do not use the HF DNA Polymerase, as it does not have terminal transferase activity.

  1. Prepare the following reaction mix on ice in a 50 μl thin-wall PCR tube:
    Component Volume
    Sheared DNA sample from Step 1 (200 ng) 10 µl
    Taq DNA Polymerase Buffer (10X) 5 µl
    dNTP Mix (50X) 1 µl
    GSP1 Primer 1 µl
    PCR-Grade Water 32.5 µl
    Taq DNA Polymerase (100X) 0.5 µl
    Total 50 µl
  1. Mix gently, and centrifuge briefly to collect droplets.
  1. Perform primer extension using the following program:
    Temperature Time
    95°C 1 minute
    62°C 5 minutes
    72°C 5 minutes
    4°C
  1. Purify the extended 3’ L1 product using the QIAquick PCR Purification Kit. Elute DNA in 30 μl of QIAGEN EB buffer.
Last modified: Mar 08, 2017

Need more help with this?
Contact Us

Thanks for your feedback.