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==Optimization of the padlock captur efficiency== The optimal dNTP0.08nM is for SNP probe with one base gap. Now I need to verify and define the optimal dNTP concentration for SNP probe with 10 bases gap. Design the optimization of the padlock capturing efficiency. set the following circularization reactions: tube 1: Probe prepared on 3/12, dNTP 0.08nM tube 2: Probe prepared on 3/12, dNTP 0.8nM tube 3: Probe prepared on 3/12, dNTP 4nM tube 4: probe CES22K (370nM), dNTP 0.08nM reaction system tube 1 tube 2 tube 3 x3 tube 4 H2O 4ul 4ul 4ul 12ul 6.7ul 10x Ampligase Buffer 1ul 1ul 1ul 3ul 1ul probe 3ul 3ul 3ul 9ul 0.3ul(CES22K,370nM) Jurkat Genomic DNA(100ng/ul) 2ul 2ul 2ul 6ul 2ul Reaction program: Tube 1: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP) -> 60C 1h<BR> -> (95c 1min -> 60C 1h) x 20cycles -> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. Tube 2: same as tube1 accept that 0.8nM dNTP Tube 3: same as tube1 accept that 4pM dNTP tube 4: same as tube1 prepartion of SLN 1ul: Storage reagents: AmpliTaq Stoffel fragment(10U/ul), AmpLigase(5U/ul),dNTP(0.5uM),dNTP(10nM),dNTP(0.2nM) *add 1ul dNTP(0.5uM) to 249ul ddH2O to get 2nM dNTP ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP 0.2ul 0.1ul 0.1ul 0.2ul (0.2nM)0.4ul 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.8nM dNTP: 0.2ul 0.1ul 0.1ul 0.2ul (2nM)0.4ul 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP: 0.2ul 0.1ul 0.1ul 0.2ul (10nM)0.4ul prepare 10x(ddH2O 6ul, Ligase Buffer 3ul, Ligase 3ul, Amplitaq 6ul, mix and aliquot into 3 tubes,<BR> then add dNTP 4ul with 0.2nM, 2nM and 10nM dNTP respectively.
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