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====gDNA==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''1000:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||3514||probes |- | DNA templet||300||ng |- | gDNA MW||1.95x10^12||g/mol |- | gDNA (300ng) ||1.5385x10^-19||mol |- | Probe (1000:1)||1.5385x10^-16||mol |- | Probe MW (3514, 150nt)||1.71585106x10^8||g/mol |- | Amount Probe req'd||26.4||ng |} *[[Matt:LabNotes/2015-5-11#Qubit_Quantification|461nM]] x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 22.5 ng/ul **Dilute to 16ul of 2.25ng/ul **10X less probes in cDNA than gDNA *Make suppressor oligo pools with 5nM and 50nM of each supp oligos **1000-fold more of each suppressor oligo: 1.5385x10^-16 mole * 1000 = 1.5385x10^-13 mole = 1.5385x10^-4 nmole = 50 nM * 3.1ul **10X less probes in cDNA than gDNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Sample Description''' | align="center" style="background:#f0f0f0;"|'''Probes''' | align="center" style="background:#f0f0f0;"|'''Target''' | align="center" style="background:#f0f0f0;"|'''1000X Supp Oligos''' | align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 1||gDNA||12||3.8||3.1 (50nM each)||3||8||30 |- | 2||cDNA dT||1.2||20||3.1 (5nM each)||3||2.7||30 |- | 3||cDNA N9||1.2||20||3.1 (5nM each)||3||2.7||30 |- | 4||NTC||1.2||20||3.1 (5nM each)||3||2.7||30 |} *Add 40ul Mineral Oil on top '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h **In BioRad Thermalcycler program says: -0.2C per cycle every 30sec * -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) * -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
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