Kun:LabNotes/Haplotyping/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) 1A 17.7 1F 11.8 1G 7.3 2E 3.1 2F 5.1
- Ligation:
End Repaired, Blunt or dA-Tailed DNA 25 μl Quick Ligation Reaction Buffer (5X) 8 μl 20 μM DNA Adaptors 2 μl Quick T4 DNA Ligase 2.5 μl H2O 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 Ind10 1F Ind11 1G Ind12 98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min. Terminated the reactions before the curves reach the plateau stage.
- The libraries were not good. See Kun:LabNotes/GenomeSeq/2010-8-26
- Repeated the library construction, and was able to make three good libraries: Kun:LabNotes/GenomeSeq/2010-9-10
- Obtained two lanes of SE 60bp reads from HL072 run.
Data file # reads GM20431_chr_1A_sequence.txt 7,613,296 GM20431_chr_1F_sequence.txt 7,760,883 GM20431_chr_1G_sequence.txt 6,546,229