Kun:LabNotes/CpgSeq/2010-6-8: Difference between revisions

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m (Created page with '==Probe prep for the DMR220k set== Library name OLID Part # DMR220k_a 0284601 G4446A DMR220k_b 0284591 G4…')
 
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   Purify the amplicons with two Qiaquick columns, eluted with 40ul EB.
   Purify the amplicons with two Qiaquick columns, eluted with 40ul EB.
===Production PCR===
===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===
===Digestion and purification===

Revision as of 21:32, 9 June 2010

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  
 Purify the amplicons with two Qiaquick columns, eluted with 40ul EB.

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