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==Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007== one reaction 3 reactions Template: 5ul each 10x Buffer: 9.5ul 28.5ul 25mM MgCl2 6ul 18ul 25mM dNTP 1.2ul 3.6ul 100uM AmpSolV6F2 0.4ul 1.2ul 100uM AmpSolV6R2 0.4ul 1.2ul Platinium Taq 1ul 3ul 50X SYBG I 0.8ul 2.4ul H2O 76.7ul 231ul 94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. [[Image: 2007-12-05-cDNA-CES22k-PCR-PAGE.png|300px]] ==Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07== Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency. <b>Reference:</b> H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM. <br> <b>Template:</b> #12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each #11-26-07: BJ cDNA 1ul #11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each Two 50ul reactions for each template. one reaction 24 reactions Template: 1ul each 10x Buffer: 5ul 120ul 25mM MgCl2 3ul 72ul 25mM dNTP 0.6ul 14.4ul 100uM AmpV6F 0.2ul 4.8ul 100uM AmpV6R 0.2ul 4.8ul iTaq 0.5ul 12ul 50X SYBG I 0.4ul 9.6ul H2O 39ul 936ul 94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Well / Set''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''C(t)''' | align="center" style="background:#f0f0f0;"|'''pM*ul''' | align="center" style="background:#f0f0f0;"|'''# molecules/ul''' |- | A2||H1(1)||10.43||0.033||19530 |- | B2||H1(1)||10.11||0.041||24888 |- | C2||H2(1)||10.55||0.030||17790 |- | D2||H2(1)||10.31||0.035||21276 |- | E2||Hues6(1)?||7.91||0.220||131760 |- | F2||Hues6(1)?||8.01||0.204||122580 |- | G2||BJ(1)?||8||0.206||123660 |- | A3||H1(2)||12.49||0.007||4075.8 |- | B3||H1(2)||12.03||0.010||5799.6 |- | C3||H2(2)||11.15||0.019||11262 |- | D3||H2(2)||12.08||0.009||5569.8 |- | E3||Hues6(2)||8.73||0.118||71040 |- | F3||Hues6(2)||8.97||0.098||59052 |- | G3||BJ(2)||12.75||0.006||3358.2 |- | H3||BJ(2)||12.28||0.008||4779 |- | |} <b>I'm not completely sure whether I added the right template to E2,F2,G2,H2.</b>
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