Kun:LabNotes/Haplotyping/2010-8-26: Difference between revisions
Jump to navigation
Jump to search
mNo edit summary |
|||
Line 24: | Line 24: | ||
2F 5.1 | 2F 5.1 | ||
*Ligation: | *Ligation: | ||
End Repaired, Blunt or dA-Tailed DNA | End Repaired, Blunt or dA-Tailed DNA 25 μl | ||
Quick Ligation Reaction Buffer (5X) 8 μl | Quick Ligation Reaction Buffer (5X) 8 μl | ||
20 μM DNA Adaptors | 20 μM DNA Adaptors 2 μl | ||
Quick T4 DNA Ligase 2.5 μl | Quick T4 DNA Ligase 2.5 μl | ||
H2O 2.5 μl | |||
Incubate in a thermal cycler for 15 minutes at 20°C. | Incubate in a thermal cycler for 15 minutes at 20°C. | ||
Purified with AMPure beads. | Purified with AMPure beads. | ||
Line 40: | Line 41: | ||
100 μl | 100 μl | ||
Barcodes 200bp | Barcodes 200bp | ||
1A | 1A Ind10 | ||
1F | 1F Ind11 | ||
1G | 1G Ind12 | ||
98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min. | 98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min. | ||
Terminated the reactions before the curves reach the plateau stage. | Terminated the reactions before the curves reach the plateau stage. |
Revision as of 15:52, 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 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.