Kun:LabNotes/GenomeSeq/2010-8-26
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Illumina multiplex library construction[edit]
- 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.
Conc(ng/ul) 300bp 800bp B.Jap-1 22.6 11.7 B.Jap-2 14.3 17.6 B.Jap-3 49.9 58.8 Ep.G-1 29.5 43.7 Ep.G-2 25.9 Ep.G-3 22.5 74.6
- Ligation:
10:1 20:1 Adaptors 300bp 800bp B.Jap-1 1.4ul 0.6ul B.Jap-2 0.9ul 0.8ul B.Jap-3 3.2ul 2.8ul Ep.G-1 1.9ul 2.1ul Ep.G-2 1.6ul Ep.G-3 1.4ul 3.5ul
End Repaired, Blunt or dA-Tailed DNA 25 μl Quick Ligation Reaction Buffer (5X) 8 μl 20 μM DNA Adaptors x μl Quick T4 DNA Ligase 2.5 μl H2O 4.5-x ul 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 300bp 800bp B.Jap-1 Ind1 Ind7 B.Jap-2 Ind2 Ind8 B.Jap-3 Ind3 Ind9 Ep.G-1 Ind4 Ind10 Ep.G-2 Ind5 Ep.G-3 Ind6 Ind12 98C 15s ->(98C 5s -> 65C 20s -> 72C 15s)x8 -> (98C 5s -> 72C 20s)x8 -> 72C 2min.
In each gel, the two lanes on the right are unamplified ligation products. File:2010-08-27-library-PCR-2.pngFile:2010-08-27-library-PCR.png
- Conclusion: library construction didn't work.
- Possibilities:
- End-repair/A-tailing reactions didn't work - I still have 30-40ul of sheared DNA
- Ligation didn't work (adaptor??) - I have ~7ul of ligation-ready DNA
- PCR didn't work - I have ~25ul of purified ligation products
- What to do next?
- Pick two samples (BJ3.3, BJ3.8), repeat the whole protocol with a new kit.
- Also repeat the ligation of BJ3.3 and BJ3.8 on the remaining ~7ul of DNA with a tube of previously made Y-adaptor.
- Change the thermal cycling protocol to
98C 30sec -> (98C 10sec -> 68C 15sec -> 72C 15sec) x 15 cycles.