Kun:LabNotes/CpgSeq/2010-6-8

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Probe prep for the DMR220k set

          Library name       OLID     Part #      
            DMR220k_a       0284601   G4446A    
            DMR220k_b       0284591   G4446A    
            DMR220k_c       0284581   G4446A    
            DMR220k_d       0284571   G4446A   
  • Resuspend the oligos in each tube with 500ul nuclease-free H2O.

Expansion PCR

                               x 4
 DMR220k_a     (20nM)          1ul                               
 DMR220k_b     (20nM)          1ul
 DMR220k_c     (20nM)          1ul                               
 DMR220k_d     (20nM)          1ul
 2x iTaq master mix:          100ul  
 100uM AP1V6:                 0.8ul
 100uM AP2V6:                 0.8ul
 H2O:                          96ul
 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 16 -> 72C 3min -> 15C hold  
 The library was slightly over-amplified. Repeated the experiment with 13 cycles.
 Purify the amplicons with four Qiaquick columns, eluted with 40ul EB.
 Yield: 20ng/ul x ~150ul = ~3ug.
 Concentration = 20ng/ul /(146bp*660Da/bp) = 208nM
 Take 20ul, add 396ul EB -> 10nM

Production PCR

   10nM 1st round amplicon        5ul
   2x Kapa SYBG qPCR Master Mix  50ul
   100uM pAP1V61U               0.4ul
   100uM AP2V6                  0.4ul
   H2O                           44ul
   ===================================
                                100ul x 16
   95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 14 -> 60C 2min -> 15C hold.
   Ethanol precipitation.

Digestion and purification