Kun:LabNotes/GenomeSeq/2010-8-27
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Repeat the Illumina library construction on B.Jap3.3 & B.Jap3.8[edit]
- End-repair:
Fragmented DNA 30 μl NEBNext End Repair Reaction Buffer (10X) 3.5 μl NEBNext End Repair Enzyme Mix 1.5 μl ----- 35 μl Incubate in a thermal cycler for 30 minutes at 20°C. Purified with MinElute columns, eluted with 15ul EB
- A-Tailing:
Blunt DNA 14.5 μl NEBNext dA-Tailing Reaction Buffer (10X) 2 μl Klenow Fragment (3´→ 5´ exo–) 1.5 μl H2O 2 μl ----- 20 μl Incubate in a thermal cycler for 30 minutes at 37°C. Purified with MinElute columns, eluted with 15ul EB Conc(ng/ul) 300bp 800bp B.Jap-3 43 56
- Ligation:
10:1 20:1 Adaptors 300bp 800bp B.Jap-3 1ul 1ul B.Jap-3(old) 1ul 1ul
End Repaired, Blunt or dA-Tailed DNA 13 μl Quick Ligation Reaction Buffer (5X) 4 μl 20 μM DNA Adaptors 1 μl Quick T4 DNA Ligase 1.5 μl H2O 0.5 ul Incubate RT for 15 minutes. 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-3 Ind3 Ind9 98C 30sec -> (98C 10sec -> 68C 15sec -> 72C 15sec) x 15 cycles. Terminated the reactions at 10th cycle. File:2010-08-28-ligation-PCR.png
- Conclusion:
- The ~120bp band still existed, indicating that it has nothing to do with end-polishing reactions and ligations.