Kun:LabNotes/Haplotyping/2010-8-26: Difference between revisions
Jump to navigation
Jump to search
m (Created page with '==Illumina multiplex library construction== *Received sheared DNA from Sara. *End-repair: Fragmented DNA 85 μl NEBNext End Repair Reaction B…') |
|||
Line 17: | Line 17: | ||
Incubate in a thermal cycler for 30 minutes at 37°C. | Incubate in a thermal cycler for 30 minutes at 37°C. | ||
Purified with AMPure beads. | Purified with AMPure beads. | ||
Conc(ng/ul) | |||
1A 17.7 | |||
1F 11.8 | |||
1G 7.3 | |||
2E 3.1 | |||
2F 5.1 | |||
*Ligation: | *Ligation: | ||
End Repaired, Blunt or dA-Tailed DNA 27 μl | End Repaired, Blunt or dA-Tailed DNA 27 μl |
Revision as of 02:03, 27 August 2010
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. 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 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.