Alice:LabNotes/2008-9-14
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extending probe length[edit]
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: 19.8ng/ul *30ul
TBU gel image of the steps: File:ZhangLab 2 2008-09-15 10hr 59min--1.jpg The spacer had high concentration around 100ng/ul, and 3ul of the spacer was loaded onto the gel. Therefore the concentration is too high, and the bands can not be distinguished.
22K probe preparation using Ribo V6[edit]
Add 40ul of KOH to the remaining product after DpnII, incubate at 94C for 1hr. The above TBU gel result showed no bands at all, which means that 94C is too high and broke up all the bonds.