Sequencing of Genomic DNA
Genomic DNA Fragmentation
Use Bioruptor.
- issolve 1 ug of genomic DNA in 300 ul of TE.
- 1 min pulse (4 C, energy level: high) x 15 cycles.
Use Covaris S2.
- Dissolve 1 ug of genomic DNA in 120 ul of TE.
- Add DNA to micro-tube, run 100 bp protocol x 2.
Add RNase A and incubate at 37 C for 30 min.
Purify DNA fragment using Qiagen minelute.
Size-selection
- 1. Run DNA on 1% Lithium Borate agarose gel with 1x SYBR-Safe at 300V.
- 2. Cut the 100 or 300 bp DNA band under blue light.
- 3. Purify DNA using Qiagen Minilute.
End-repair
- 4. Assemble the following reaction mix.
| DNA | 20 ul |
| 10 x T4 Ligation Buffer | 5 ul |
| 10 mM dNTP mix | 1 ul |
| T4 PNK (NEB) | 2 ul |
| T4 DNA Pol(NEB) | 2 ul |
| H2O | 20 ul |
- 5. Incubate at 20 C for 30 min.
- 6. Purify using AMPure XP beads.
dA-tailing
- 7. Prepare the reaction as follow:
| DNA | 20 ul |
| 10 x T4 Ligation Buffer | 5 ul |
| 10 mM dNTP mix | 1 ul |
| T4 PNK (NEB) | 2 ul |
- 8. Incubate at 37C for 30 min.
- 9. Purify DNA using AMPure XP.
Adapter ligation
- 10. Assemble ligation reaction on ice as follow:
| DNA | 20 ul |
| PE adapters (50 uM) | 1 ul |
| 2x Ligation Buffer (Enzymatics) | 25ul |
| T4 DNA Ligase (Enzymatics) | 3 ul |
- 11. Incubate at 20C for 15 min.
- 12. Purify DNA using AMPure XP.
PCR amplification
- 13. Assemble PCR reaction on ice as follow:
| Purified ligated gDNA | 10 ul |
| PE Primer (S-oligo, 10 uM each) | 1 ul |
| 5x Phusion HF Buffer | 5ul |
| 10 mM dNTP | 1 ul |
| H2O | to 25 ul |
| Phusion II (NEB) | 1 ul |
- 14. PCR for 8 cycles and purify library using AMPure XP.
- 15. Check the quality of library on Agilent Bioanalyzer.
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