Alice:LabNotes/2008-11-4
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diagnosing spacer annealing and insertion efficiency
For the product and procedure from previous steps, please follow this link (10/14/08 LabNote): [[1]]
Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM the final concentration of spacer is 20nM, so 20nM x 50ul = 100nM x amount needed(ul) Add 10ul of each primer, denature at 95C for 5min, and then anneal at 65C for 15 min. Add the following reagents along with the spacers then denature at 95C for 5 min, anneal at 65c for 15 min. template (1) 680nM 1.5 ul spacer mix (100nM each) 20 ul 10x Ampligase Buffer 5 ul Ampligase(5U/ul) 1 ul ddH2O 15.6 ul ------------------------------------------ total 50 ul Add 1ul of dNTP, 0.5ul of Stoffel fragment to the reaction system above, 60C for 20min. File:ZhangLab 2 2008-11-04 16hr 33min.jpg TBE gel result: 1: product from reaction system above(after spacer insertion) 2: cosmic probe product from PCR