Kun:LabNotes/GenomeSeq/2010-9-9
Jump to navigation
Jump to search
Repeat the Illumina library construction on the remaining DNA[edit]
- In this experiment, I used the adaptors that Alice prepared. I also reduce the AMPure beads to 1 volume of the reaction (eliminated small DNA fragments).
- 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 Qiaquick columns, eluted with 30ul EB Concentration (ng/ul) BJ1.3 28 BJ1.8 16 BJ2.3 20 BJ2.8 19 EP1.3 27 EP1.8 35 EP2.3 26 EP3.3 27 EP3.8 38 1A 16 1G 13 1F 12
- A-Tailing:
Blunt DNA 28 μl NEBNext dA-Tailing Reaction Buffer (10X) 3.5 μl Klenow Fragment (3´→ 5´ exo–) 2.5 μl H2O 1 μl ----- 35 μl Incubate in a thermal cycler for 30 minutes at 37°C. Purified with MinElute columns, eluted with 20ul H2O. Concentration (ng/ul) BJ1.3 20 BJ1.8 12 BJ2.3 17 BJ2.8 16 EP1.3 28 EP1.8 35 EP2.3 30 EP3.3 24 EP3.8 51 1A 11 1G 6 1F 6
- Ligation:
Adaptors (20uM) adaptor:insert=1:20 BJ1.3 2.0 BJ1.8 0.5 BJ2.3 1.7 BJ2.8 0.6 EP1.3 2.8 EP1.8 1.3 EP2.3 3.0 EP3.3 2.4 EP3.8 2.0 1A 1.1 1G 0.6 1F 0.6
End Repaired, Blunt or dA-Tailed DNA 18 μl Quick Ligation Reaction Buffer (5X) 6 μl 20 μM DNA Adaptors x μl Quick T4 DNA Ligase 2 μl H2O 4-x ul Incubate RT for 15 minutes. Purified with 1V AMPure beads.
- PCR:
Adapter ligased DNA 10 μl PCR_Fx(100uM) 0.2 μl PCR_R(10uM) 2 μl 50X SYBR Green I 0.8 μl H2O 37 μl 2X Phusion HF MM 50 μl ----- 100 μl 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles. -> (98C 10sec -> 72C 20sec) x 4 cycles. File:2010-09-09-ligation-PCR1.png File:2010-09-09-ligation-PCR2.png
- Conclusion: All these libraries worked, indicating that somehow the adaptors I used in the previous experiments are problematic.