Kun:LabNotes/Haplotyping/2010-8-26
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Illumina multiplex library construction
- Received sheared DNA from Sara.
- End-repair:
Fragmented DNA 85 μl NEBNext End Repair Reaction Buffer (10X) 10 μl NEBNext End Repair Enzyme Mix 5 μl ----- 100 μl Incubate in a thermal cycler for 30 minutes at 20°C. Purified with AMPure beads.
- A-Tailing:
Blunt DNA 42 μl NEBNext dA-Tailing Reaction Buffer (10X) 5 μl Klenow Fragment (3´→ 5´ exo–) 3 μl ----- 50 μl Incubate in a thermal cycler for 30 minutes at 37°C. Purified with AMPure beads.
- Ligation:
End Repaired, Blunt or dA-Tailed DNA 27 μl Quick Ligation Reaction Buffer (5X) 8 μl 20 μM DNA Adaptors 2.5 μl Quick T4 DNA Ligase 2.5 μl Incubate in a thermal cycler for 15 minutes at 20°C. Purified with AMPure beads.
- PCR:
Adapter ligased DNA 10 μl PCR_F(10uM) 2 μl PCR_R.N2IndX(10uM) 2 μl 50X SYBR Green I 0.8 μl H2O 37 μl 2X Phusion HF MM 50 μl ----- 100 μl Barcodes 200bp 1A Ind8 1F Ind9 1G Ind10 2E Ind11 2F Ind12 98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min. Terminated the reactions before the curves reach the plateau stage.