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.