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 41ul EB (total vol: 12 x 41 = 492ul).
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/ulx492ul= 95.5ug 192.7ng/ulx492ul = 94.8ug
Lambda exo digestion
DMR220k_S4 DMR220k_S5 Qiaquick (QQ)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 Total amount of QQ amplified amplicon 94.4ug 93.6ug - 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) 81.3 75.9 O.D.260/280 1.96 1.96 Yield: conc. x total vol. 81.3ng/ulx240ul = 19.5ug 75.9ng/ulx240ul = 18.2ug
Removal of amplification adaptors
DMR220k_S4 DMR220k_S5 ss-amplicon 75ul (6.1ug) 75ul (5.7ug) USER 5ul 5ul ----------------------------------------------------- Total 80ul x 3 80ul x 3 Total amount of ss amplicon 18.3ug 17.1ug - 37C 1hr - Add 10x DpnII buffer 10ul 10ul 100uM RE-DpnII-V66 guide oligo 5ul 5ul - 94C 2min, 37C 3min - Add DpnII 5ul 5 ul - 37C 3hr - Add USER 5ul 5ul 10x DpnII buffer 10ul 10ul H2O 85ul 85ul - 37C O/N