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== PCR amplification after insertion of spacer_107bp 2008-08-14== reaction system x2 H2O 22.4ul 44.8 iProof 2x mix 25ul 50 Ap1V4U(100uM) 0.2ul 0.4 Ap2V4(100uM) 0.2ul 0.4 50x SYBG I 0.2ul 0.4 template 2ul Total 50ul 100 98C 2min -> 22 cycles of (98C 10sec -> 63C 20sec -> 72C 20sec) -> 72C 3min -> 4C [[Image:20080814_PCR after insertion of spacer.jpg]]20080814_PCR after insertion of spacer I realized that the probe, after the Stoffel fragment amplification and ligation of cirlce helper, will be converted into a <BR>double-strand cirlce that has two ALuI restriction site(one is on linker and the other is on cirlchelper). The following AluI digestion will cut <BR>the probe at two sites and produce a 90-mer and a 70-mer pieces. The 160-mer on the gel is the <BR>reverse strand which was cut by AluI on the circlhelper site. To confirm the hypothesis, I digested the circularized cpg30k_V2.2 (with circlehelper) by AluI without AluI-oligo. [[Image:20080817-digestion without AluI oligo.jpg]] The image showed that the AluI digested the circle without AluI-oligo, which means that the circularization with circlehelper may form a double-stranded circular DNA (180-mer cut from TBU gel). <BR> The digested pattern looks the same as that with AluI-oligo. I ordered a new circlehelper_V4.2_2_Ida, which replaced the AluI site with 'ATCA': 5'- AAC AGT GCT CTT CCA GTC TAC ATC AGA ATT CTA GCC TCA TGC GTA TCC GAT C -3'
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