Alice:LabNotes/2008-9-14
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extending probe length
PCR after circlehelper insertion: H2O 40ul SYBR green (50x) 0.4ul Forward primer AmpRV6.2_2(100uM) 0.2ul Reverse primer AmpFV6.2_2(100uM) 0.2ul Taq master mix(2x) 50ul probe template (product after Exo I&III) 10ul 94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold T7 exonuclease digestion: Add 2ul of T7 exonuclease to the PCR product 25C 2hr -> 4C Purify with Qiaquick columns, and measure the concentration using Nanodrop to determine the amount of spacer needed. Nanodrop result:
22K probe preparation using Ribo V6
Add 40ul of KOH to the remaining product after DpnII, incubate at 94C for 1hr.