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==Experiment: Extending Cosmic Probe Length== ==PCR amplification of cosmic probe== PCR reaction system x48 H2O 50ul 2400ul SYBR green (50x) 0.4ul 19.2ul primer mix (100uM) 0.2ul 9.6ul Taq master mix(2x) 50ul 2400ul probe template (cosmic) 0.05ul 2.4ul 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold Purify the product using ethanol purification: 1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, 6 mL 100% Ethanol. 2. Store at -70C for more than 20 min. 3. Spin at 4000rpm for 15 min at 4C. 4. Remove all liqid, you should see a blue pallette at the bottom of the tube. 5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube. 6. Spin at 10000 rpm for 5 min at 4C. 7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 100 uL dH2O. ==T7 exonuclease digestion (10/7/08)== 1. The tubes were purified using ethanol purification, and diluted in 100ul H2O for every 24 reactions. 2. Add ratio of 0.5ul/tube amount of T7, which is 12ul of T7 and 11ul of NEBuffer #4 to each tube(24 reactions). 3. 25C for 2 hrs. 4. Qiaquick column purification, elute in 30ul of H2O. 5. Measure with Nanodrop. Nanodrop result: 24.2ng/ul * 35ul ==Circularization reaction== the concentration of the template from last step is 24.2 ng/ul, which is 24.2/145/330= 509nM reaction system 1: reaction system 2: template (509nM) 2ul template (509nM) 1ul 10x Ampligase Buffer 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 1ul CircleHelper V4.2(1uM) 2ul dNTP(10mM) 0.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 14.5ul ddH2O 15ul 94C 2 min -> 55C 20 min [[Image:ZhangLab_2 2008-10-08 15hr 28min.jpg]] result: the band at 100bp has much higher intensity than all other bands, however, it is not the band we expected. Also, few more experiments will be done to determine the content of this 100bp band before I continue with the next step. add 1.5ul Exo I &III + 10ul of product from reaction system 1 37C 2hr -> 95 10min -> 4C Purify using Minelute columns ==circularization under different reaction system (10/9/08)== reaction system: 1 2 3 4 5 template + + + - + circleHelper + + + + - Ligase + - + + + Stoffel fragment + + - + + template (509nM) 1ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 2ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 15ul 94C 2 min -> 55C 20 min [[Image:ZhangLab_2 2008-10-09 15hr 52min.jpg]] result: the primer of the first PCR step is not phosphorylated at the 5'end, therefore the ligase would not be able to work. The gel above showed very interesting results, whereas looks like the template and the stoffel fragment are having some unspecific activities. However, I am not entirely sure what happened within each reaction, especially with the 100bp band. The steps will be repeated using phosphorylated primer, and the same reaction system will be used again later to compare the results. ==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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