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==== Probe & buffer reaction mix ==== * I diluted 10x of V1N3 by mixing 8ul of V1N3 with 72ul H2O and re-quantified concentration with Qubit ssDNA assay: <u>0.73ng/ul</u> *'''Mix XX = amount of V1N3 in each reaction''' :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.22''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||0.30||1.20 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||5.30|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.44''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''6x''' |- | Diluted V1N3||0.60||3.60 |- | 10X Ampligase Buffer||2.00||12.00 |- | H2O||3.00||18.00 |- | Sum||5.60|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 0.88''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||1.20||4.80 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||6.20|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 1.1''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||1.50||6.00 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||6.50|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 2.2''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''6X''' |- | Diluted V1N3||3.01||18.06 |- | 10X Ampligase Buffer||2.00||12.00 |- | H2O||3.00||18.00 |- | Sum||8.01|| |} :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Mix 4.4''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''4x''' |- | Diluted V1N3||6.02||24.08 |- | 10X Ampligase Buffer||2.00||8.00 |- | H2O||3.00||12.00 |- | Sum||11.02|| |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 2ul SLN mix (2U/ul Hemo KlenTaq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 19h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 4C hold.
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