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==diagnosing spacer annealing and insertion efficiency== <font color=green>For the product and procedure from previous steps, please follow this link (10/14/08 LabNote): [[http://genome-tech.ucsd.edu/LabNotes/index.php/Alice:LabNotes/2008-10-14]]</font> 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 (actually 211nM) 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. <----unnecessary [[Image: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 Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min ==PCR amplification after spacer insertion (11/6/08)== H2O 40ul SYBG 0.4ul primer mix (AP1V4IU+AP2V4) 0.2ul Taq master mix 50ul Template (from previous step) 10ul ---------------------------------------- total 100ul TBE gel result: [[Image:ZhangLab_2 2008-11-06 16hr 17min.jpg]] 1: PCR product of cosmic probe 2: PCR product after spacer insertion Conclusion: The TBE gel result of the PCR product after spacer insertion showed two clear bands, one having the same size as lane 1, and the other band having extra 60bp in length. This shows that the spacer is successfully inserted into the template.
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