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==Circularization== Equation: final volume x final concentration = initial volume x initial concentration the final concentration of template is 20nM, so 20nM x 30ul = 956nM x amount we need, which is 0.623 ul. CircleHelper V_short is 100uM, so we take 1 ul of it and add 99ul of H2O to dilute it 100 times to 1uM. The final concentration of circlehelper is 60nM, so we need 1.8ul. Add the ligase at 0.02 units/ul ratio. since the total volume is 30ul, we will need to add 0.12 ul of ampligase. T4 ligase is 2000U/ul. so we will dilute it to 5U/ul, which is to add 0.5ul of ligase to 199.5ul of H2O. reaction system 1: template (956nM) 0.6ul 10x Ampligase Buffer 3ul CircleHelper V_short(1uM) 1.8ul Ampligase(5U/ul) 0.12ul ddH2O 27ul ----------------------------------------- total 30ul 94C 2 min -> 55C 20 min reaction system 2: template (956nM) 0.6ul 10x T4 ligase Buffer 3ul CircleHelper V_short(1uM) 1.8ul T4 ligase(5U/ul) 0.12ul ddH2O 27ul ----------------------------------------- total 30ul 94C 2 min -> 25C 20 min add 2ul Exo I &III 37C 2hr -> 95 10min -> 4C Purify using Minelute columns TBU gel result: [[Image:ZhangLab_2 2008-10-15 15hr 32min.jpg]] ==PCR after circlehelper insertion(10/15/08)== reaction systems: 1: circularization of system 1 product from above (before exo I&III digestion) 2: circularization of system 2 product from above (before exo I&III digestion) 3: circularization of system 1 product from above (after exo I&III digestion) 4: circularization of system 1 product from above (after exo I&III digestion) 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 TBE gel result: [[Image:ZhangLab_2 2008-10-16 13hr 28min.jpg]] lane 1-4 are the product from reaction systems above, lane 5 is the 10bp ladder ==repeat circularization(10/16/08)== reaction system 1: template (956nM) 0.6ul 10x Ampligase Buffer 3ul CircleHelper V_short(1uM) 1.2ul Ampligase(5U/ul) 1ul ddH2O 27ul ----------------------------------------- total 32.8ul 94C 2 min -> 55C 20 min reaction system 2: template (956nM) 0.6ul 10x T4 ligase Buffer 3ul CircleHelper V_short(1uM) 1.2ul T4 ligase(5U/ul) 1ul ddH2O 27ul ----------------------------------------- total 32.8ul 94C 2 min -> 25C 20 min Purify the products using qiaquick columns. ==Repeat 2: Extending cosmic probe (10/16/08)== PCR amplification (8 tubes total) H2O 50ul SYBR green (50x) 0.4ul primer mix (100uM) 0.2ul Taq master mix(2x) 50ul probe template (cosmic) 0.05ul 94c 2min -> 94c 30sec -> 60C 45sec-> 72C 45sec-> Plate read -> 20 cycles -> 72C 5min -> 15C hold Purify two tubes of the product using Qiaquick column, and elute the DNA in 30ul ddH2O. Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, and 2ul of Lambda Exo. Purify it using Qiaquick columns again. Nanodrop result: 52.2ng/ul, which is 1.1uM. ==Repeat 2: circularization (10/17/08)== reaction system 1: template (956nM) 0.6ul 10x Ampligase Buffer 3ul CircleHelper V_short(1uM) 1.2ul Ampligase(5U/ul) 1ul ddH2O 27ul ----------------------------------------- total 32.8ul 94C 2 min -> 55C 20 min reaction system 2: template (956nM) 0.6ul 10x T4 ligase Buffer 3ul CircleHelper V_short(1uM) 1.2ul T4 ligase(5U/ul) 1ul ddH2O 27ul ----------------------------------------- total 32.8ul 94C 2 min -> 25C 20 min Add 2ul of Exo I&III to the two products -> 37C 2hr -> 95C 10min ==Repeat 2: PCR after circlehelper insertion(10/21/08)== systems setup: 1: product of repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III 2: product of repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III 3: product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III 4: product of Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III 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)* 22 cycles -> 72C 5min -> 15C hold ==Gel results of the experiments under different conditions (10/20-21):== TBU gel result of the circularization products (see above for protocol used): [[Image:ZhangLab_2 2008-10-20 11hr 04min.jpg]] 1: repeat 2-PCR product of cosmic probe 2: repeat 2-digested PCR product using lambda Exo 3: repeat circularization(10/16/08)-reaction system 1 4: repeat circularization(10/16/08)-reaction system 2 5: repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III 6: repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III 7: Repeat 2: circularization (10/17/08)-reaction system 1 8: Repeat 2: circularization (10/17/08)-reaction system 2 9: Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III 10: Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III TBE gel of the PCR results: [[Image:ZhangLab_2 2008-10-21 15hr 37min.jpg]] (see sections <font color="green"> Repeat 2: Extending cosmic probe (10/16/08)</font> and <font color="green"> Repeat 2: PCR after circlehelper insertion(10/21/08) </font>above for detailed protocol) 1: PCR product of repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III 2: PCR product of repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III 3: PCR product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III 4: PCR product of Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III 5: PCr product of Repeat 2: Extending cosmic probe (10/16/08) <Font Color="green">Note: the PCR curve never shoot up for lane 2 and 4. Lane 3 was digested with lambda Exonuclease and shoot up at 10th cycle, while lane 1 was treated with T7 exonuclease and shoot up around 15th cycle. </Font>
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