Alice:LabNotes/2009-10-9
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iPS library construction
- Received five sheared samples from Billy, ~400ng in ~80ul each
- Use the NEBNext DNA Sample Prep Master Mix Set I kit for library construction.
- Use the Y-adaptor for ligation.
End repair
DNA ~50ul End Repair 10X buffer 10ul End Repair Enzyme Mix 5ul H2O ~30ul Total 100ul Room temperature for 20min, purified with Qiaquick column, eluted with 39ul EB
A Tailing
End repaired DNA 37ul dA-Tailing buffer (10x) 5ul Klenow exo- 3ul H2O 5ul Incubate at 37C for 30min
- Purify with Qiaquick columns, eluted with 30ul EB.
- Perform size selection on 20ul DNA with 2% size selection gel, extract the ~100bp bands.
Ligation
- Mix 20ul A_adaptor_b (100uM), 20ul A_adaptor_t (100uM), 5ul 10x Taq buffer, 55ul H2O.
- 94C 3min -> ramp to 20C at 0.1C/sec -> 4C hold.
- Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors.
- Include a positive control.
A-tailed DNA 15ul 5X QuickLigase buffer 10ul 20uM Adaptor mix 5ul Quick T4 ligase 5ul H2O 15ul RT for 15min Purified with MinElute columns, eluted with 15ul EB.
PCR
- Check the size distribution of the PCR products with 1ul of ligation products first
Template 1ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 0.4ul Solexa_PCR_lo_PE (10uM) 0.4ul H2O 49ul 50X SYBG I 0.4ul 98C 30sec -> 13 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) File:ZhangLab 2 2009-10-09 15hr 40min.jpg