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====CircLigase II test to prove what is circularized DNA==== =====Sample set===== *Run CircLigase II reaction **input ssDNA : Custom ssDNA (50bp) having adapter sequence **+CircLigaseII and -CircLigase II **Reaction condition : 60C Thermo Cycler for 1hr 30min. --> 80C for 10min =====Reaction condition===== *CircLigase II : 20uL total reaction volume {| {{table}} | Reagent || | Conc. || | Volume || | Final Conc. |- |ss DNA || |2uM || |5uL || |0.5uM |- |10x Reaction Buffer || |10x || |2uL || |1x |- |MnCl2 || |50mM || |1uL || |2.5mM |- |Betain || |5M || |4uL || |1M |- |CircLigase II || |100U/uL || |1uL || |5U/uL |- |H2O || | || |7uL || | |} *Run Exo I reaction **9uL from CircLigase and CircLigase II reaction **add 1uL of Exo I **Run 37C for 1hr. --> 80C for 15min =====Result===== *TBU gel (All samples were denatured by 75C for 8min) *Result **-CircLigase II showed the same band to Custom ssDNA, **and Exo I reaction of –CircLigase II showed nothing (Lane 7). **Thus, the band in +CircLigase II and Exo I reaction of +CircLigase II are circularized DNA. **In result, circularized 50bp of custom ssDNA (CircAdapt2 Primer) runs faster than linear one. **This results are consistent with the previous results. **RCA reaction is running with two samples, +CircLigase II and Exo I of +CircLigase II. *Gel table {| {{table}} | #1 || | #3 || | #4 || | #5 || | #6 || | #7 || |- | 10bp || | Custom ssDNA || | +CircLigase II || | Exo I of +CircLigase II || | -CircLigase II || | Exo I of -CircLigase II |} *[[File:Gel_CircII_Adapt2_2b.jpg|350px]]
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