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==<b>Prepare Spacer</b>== we have two spacer that are waiting to be tested, one is 100 bp FR and other one is 300 bp FR. PCR reaction system setup: x12 H2O 80.1ul 961.2 ul 10x Stoffel buffer 10ul 120 ul MgCl2(25mM) 6ul 72 ul dNTP(10mM each, NEB) 2ul 24 ul primer (Forward) 0.4ul 4.8 ul primer (Reverse) 0.4ul 4.8 ul 50x SYBG I 0.4ul 4.8ul template ( few colonies picked from E coli plate ) Jumpstart Taq 0.6ul 7.2 ul 94c 2min -> 10 cycles (94c 45sec -> 60C 45sec -> 72C 45sec-> Plate read) -> 10 cycles ( 94C 45 sec -> 68C 45 sec -> 72C 45 sec -> plate read) -> 72C 5min -> 15C ethanol precipitation and dilute in 125ul ddH2O. Add 15ul 10x Lambda Exonuclease buffer and 8ul Lambda nuclease(5K/ml). 37C 2h. Quaquick column purification. Elute in 30ul ddH2O. Nanodrop result: 100bp spacer: 155.5ng/ul*30ul= 4665 ng tube 2: 163.3ng/ul*30ul= 4899 ng 300bp spacer: 237.8ng/ul*30ul= 7134 ng tube 2: 240.6ng/ul*30ul= 7218 ng Result: TBE gel of the spacer after PCR: TBU gel of the products (8-5-08): [[Image:ZhangLab_2 2008-07-30 09hr 36min.jpg]] [[Image:ZhangLab_2 2008-08-05 18hr 22min-1.jpeg]]
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