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.
- 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
- Mix gently, and centrifuge briefly to collect droplets.
- Perform primer extension using the following program:
Temperature Time 95°C 1 minute 62°C 5 minutes 72°C 5 minutes 4°C ∞
- Purify the extended 3’ L1 product using the QIAquick PCR Purification Kit. Elute DNA in 30 μl of QIAGEN EB buffer.
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