Alice:LabNotes/2009-10-9

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Revision as of 23:29, 9 October 2009 by >Zsakura2 (New page: ==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-adap...)
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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

  1. 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