Noi:LabNotes/2010-10-21
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Probe prep for the DMR220k S4 and S5
(followed Dr. Zhang's conditions)
Production PCR
10nM 1st round amplicon 0.2ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul H2O 49ul =================================== 100ul x 96
95C 30sec (95C 3sec -> 55C 30sec-> 60C 20sec) x 15 cycles 60C 2min 15C hold. Ethanol precipitation; re-purified with twelve Qiaquick columns, each eluted with 40ul EB (total vol: 12 x 40 = 480ul).
DMR220k_S4 DMR220k_S5 Amplified amplicon conc. (ng/ul) 194.2 192.7 O.D.260/280 1.93 1.92 Yield: conc. x total vol. 194.2ng/ulx480ul= 93.2ug 192.7ng/ulx480ul = 92.5ug
Lambda exo digestion
DMR220k_S4 DMR220k_S5 Qiaquick amplified amplicon 108ul (21.0ug) 108ul (20.8ug) 10x Lambda Exo buffer 15ul 15ul Lambda exo 10ul 10ul H20 17ul 17ul ============================================================ Total 100ul x 4.5 100ul x 4.5
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).
DMR220k_S4 DMR220k_S5 Single-strand(SS) amplicon conc. (ng/ul) O.D.260/280 Yield: conc. x total vol.