Kun:LabNotes/HiResChrPaint/2012-4-4

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Processing of oligo pools (STEP 1)

  • Received the oligo pools from Mycorarray in four tubes. These are "ssDNA from emulsion PCR pool". Resuspend each pool in 500ul nuclease free H2O.
  • Take 50ul of each oligo pool, dilute to 20nM. These will be the working tubes.
   5431_ssDNA  80k_set_4   ~2.8ug   85pmole     170nM      50ul+374ul H2O => 20nM
   5432_ssDNA  80k_set_1   ~1.8ug   55pmole     109nM      50ul+223ul H2O => 20nM
   5433_ssDNA  80k_set_2   ~1.7ug   52pmole     103nM      50ul+208ul H2O => 20nM
   5435_ssDNA  80k_set_3   ~2.8ug   85pmole     170nM      50ul+374ul H2O => 20nM
  • There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR.

Production PCR

   V6 pool for all four sets
                                Each     x16
   20nM oligo pool              0.2ul    3.2
   2x Kapa SYBG qPCR Master Mix  50ul    800
   100uM pAP1V61U               0.4ul    6.4
   100uM AP2V6                  0.4ul    6.4
   H2O                           44ul    704
   ===================================
                                100ul 
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
   V4 pool for sets #1, #3, #4
                                Each     x16
   20nM oligo pool              0.2ul    3.2
   2x Kapa SYBG qPCR Master Mix  50ul    800
   100uM pAP1V41U               0.4ul    6.4
   100uM AP2V4                  0.4ul    6.4
   H2O                           44ul    704
   ===================================
                                100ul x 16
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.