This procedure can be used to validate the structure of the amplified fragments and ensure they contain intact P5/P7 sequences for Illumina cluster generation, correct adapter sequences, as well as defined mobile element-chromosome junction regions.
- Prepare the following reaction mix on ice in a 50 μl thin-wall PCR tube:
Component Volume DNA sample from 3rd PCR (Step 6) 2 µl Taq DNA Polymerase Buffer (10X) 5 µl dNTP Mix (50X) 1 µl P7 Indexed Primer 1 (as appropriate) 1 µl P5 Primer 1 µl PCR-Grade Water 39 µl Taq DNA Polymerase (100X) 0.5 µl Total 50 µl
- Mix gently and centrifuge briefly.
- Amplify using the following PCR program:
98°C, 60 seconds 1 cycle 98°C, 15 seconds
62°C, 15 seconds
72°C, 30 seconds
5 cycles 72°C, 60 seconds 1 cycle 4°C, ∞ 1 cycle
- Follow the instructions in the TA PCR Cloning Kit (Invitrogen/ThermoFisher) to clone 1 µl of the PCR reaction into the TA cloning vector and transform the TA cloning mix into the appropriate bacterial strain.
- Pick 20 colonies at random from the plate. For colony PCR, use the T3/T7 PCR protocol. Sequence the 20 random colonies with T3/T7 primers. If the 3’ L1 DNA was prepared correctly, the amplified fragments should contain intact P5 and P7 sequences for Illumina cluster generation and correct adapter sequences, as well as defined mobile element-chromosome junction regions as shown on Figure 2.
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