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==Repeat circularization steps== <b>PCR reaction system </b> H2O 50ul SYBR green (50x) 0.4ul primer AP1V4(100uM) (phosphorylated) 0.2ul primer AP2V4(100uM) 0.2ul Taq master mix(2x) 50ul probe template (cosmic) 0.05ul 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> <b>T7 exonuclease digestion:</b> Add ratio of 0.5ul/tube amount of T7 25C for 2 hrs. (Only two out of the 10 tubes prepared is used for purification and later steps) Qiaquick column purification, elute in 30ul of H2O. Measure with Nanodrop. Result: 45.4ng/ul <b>Circularization reaction: (10/10/08)</b> the concentration of the template from last step is 45.4ng/ul, which is 956nM. reaction system: 1 2 3 4 5 template + + + - + circleHelper + + + + - Ligase + - + + + Stoffel fragment + + - + + template (956nM) 1ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 1ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 15ul 94C 2 min -> 55C 20 min add 2ul Exo I &III to the product after Exo I&III 37C 2hr -> 95 10min -> 4C Purify using Minelute columns TBU gel of the products: [[Image:ZhangLab_2 2008-10-10 15hr 39min-.jpg]] <b>PCR after circlehelper insertion:</b> 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
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