Kun:LabNotes/ExonomeSeq/2008-1-14

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Making sequencing libraries from the 250bp bands purified from 01/09/08 exp[edit]

DNA synthesis with random hexamers[edit]

                           N6
     DNA                  20ul
     10X Klenow buffer     3ul
     1mM N6              0.6ul
     10mM dNTP           1.2ul
     H2O                 2.2ul

94C 2min -> 16C 1min -> add 1.5ul 20U/ul Klenow exo- -> 16C 10min -> 37C 5min -> 94C 1min -> 16C 1min -> add 1.5ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> purified with MinElute columns.

USER enzyme digestion[edit]

Add 2ul USER enzyme to 20ul purified DNA in dH2O -> 37C 1h -> purified with MinElute columns.

Ligation to Solexa adaptors[edit]

  • Mix 100ul of Solexa_1_up and Solexa_1_lo_noP adaptors (200uM) with 200ul 1X PCR buffer, the final concentration is 50uM.
  • Mix 100ul of Solexa_2_up and Solexa_2_lo_noP adaptors (200uM) with 200ul 1X PCR buffer, the final concentration is 50uM.
  • 95C 5min -> turn of the program, let the tube slowly cold down in ~10 minute -> transfer on ice.
                        N6 primed         USER digested
      DNA                  13ul                 22ul
  50uM Solexa_1             1ul                  1ul
  50uM Solexa_2             1ul                  1ul
  2X Quick ligation buffer 25ul                 25ul
  Quick ligase              1ul                  1ul
  H2O                       9ul                  0ul
  mix well -> room temperature for 20min -> 75C 10min
  
  Run 20ul in 4% clonewell gel, collect the fraction of 100~150bp. 
  Purify the remaining 20ul reactions with MinElute columns.

Nick translation[edit]

  DNA                    15ul
  10X ThermoPol Buffer    2ul
  10mM dNTP             0.4ul
  1mg/ml BSA              2ul
  8U/ul Bst pol           1ul
  65C 25min -> place on ice

PCR enrichment of ligated DNA[edit]

    Nick-translated DNA      5ul
    2X iProof master mix    25ul
    100uM Solexa_PCR_up   0.25ul
    100uM Solexa_PCR_lo   0.25ul
    H2O                   19.3ul
    50X SYBG I             0.2ul
    98C 30sec -> 18 cycles of (98C 10sec -> 68C 30sec) -> 72C 3min ->15C hold
    
   File:2008-01-17-E55kv1-SolLib-PCR.png