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==spacer insertion== <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> template used: (1) PCR product of repeat: circularization(10/16/08)-reaction system 1 after treated with Exo I&III (2) PCR product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III PCR results from lane 1 and 3 (see section Repeat 2: PCR after circlehelper insertion(10/21/08) from link above) was purified with Qiaquick columns,elute DNA in 30ul ddH2O. Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, 2ul of Lambda Exo. 37C 2hrs -> 90C 10 min Purify it using Qiaquick columns again and use Nanodrop to test the concentration. Nanodrop result: (1) 5.7 ng/ul = 119nM (2) 10.1 ng/ul = 680 nM <font color=red><-this is a calculation error!</font> Note: this proves that digest the PCR product with exonuclease gives about twice as much %yield as using T7 exonuclease. Equation: final volume x final concentration = initial volume x initial concentration *previous experiments show that ligation process works better with low concentration of template and primers* The amount of insertion spacer oligo ordered was 2nmol, and the reverse arm was 4.8nmol. Diluting them using 200ul and 480ul of ddH2O respectively yields final concentration of 10uM. Dilute the spacer to 10nM since the PCR product concentration is quite low (we need 1:1 ratio of spacer:template) Prepare the spacer: 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 10min. Prepare the template: the final concentration of template is 20nM, so 20nM x 50ul = template(nM) x amount needed(ul) template (1) needs 20nM x 50ul = 119 nM x <font color=green>8.4ul</font> template (2) needs 20nM x 50ul = 680 nM x <font color=green>1.5ul</font> Add 8.4ul of template(1) and 14.1ul of ddH2O; Add 1.5ul of template(2) and 21ul of ddH2O; denature at 95C for 5min, quickly add the spacer prepared from last step, anneal at 65C for 10 min Add the remaining reagent in the chart below (color green) and anneal at 60C for 20min. spacer insertion: reaction system 1: template (1) 119nM 8.4 ul spacer mix (100nM each) 20 ul <font color=green> 10x Ampligase Buffer 5 ul Ampligase(5U/ul) 1 ul dNTP(10mM) 1 ul Stoffel fragment(10U/ul) 0.5 ul</font> ddH2O 14.1 ul ------------------------------------------ total 50 ul reaction system 2: template (2) 680nM (211nM actually) 1.5 ul spacer mix (100nM each) 20 ul<font color=green> 10x Ampligase Buffer 5 ul Ampligase(5U/ul) 1 ul dNTP(10mM) 1 ul Stoffel fragment(10U/ul) 0.5 ul</font> ddH2O 21 ul ------------------------------------------ total 50 ul Add 2ul of Exo I&III -> 37C 2hr -> 95C 10min purify the products with Qiaquick columns [[Image:ZhangLab_2 2008-10-31 13hr 37min.jpg]] 1: template (1) before spacer insertion 2: template (2) before spacer insertion 3: reaction system 1 after spacer insertion 4: reaction system 2 after spacer insertion 5: reaction system 1 after spacer insertion and ExoI&III 6: reaction system 2 after spacer insertion and ExoI&III PCR amplification after spacer insertion (10/30/08): H2O 40ul Primer mix 100uM (Ap1V4IU +Ap2V4) 0.2ul 50x SYBG I 0.4ul template(from last step) 10ul Taq 2x master mix 50ul 94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x25 cycles -> 72C 5min -> 15C hold [[Image:ZhangLab_2 2008-10-31 14hr 03min.jpg]] 1: PCR product of cosmic probe 2: reaction system (1) after PCR 3: reaction system (2) after PCR ==Diagnosing spacer insertion efficiency (10/31/08)== The above gel images seems somehow problematic before the cosmic probe product looks weird, it should have bands around 145bp region. Therefore I am going to repeat and procedures and test if the spacer insertions are able to anneal with each other. Then put them into the template using the same protocol as Circlehelper insertion. 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 20min. several results of the TBE gel from above system had problems such as bad gel or not loading enough samples, therefore they are not posted.
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