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===20gap Probe Capture Plan=== *From [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22] and [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-29]: ~37.4 pmol (2.35 ug) in 140ul = 16.79ng/ul(Combined on 6-6-2013 into single tube) *Use HAPMAP (ID C1) gDNA (87ng/ul) as template/target *Use a probe:target ratio of 100:1 *Hosuk wants to use some circularized oligos to generate rolonies *Hosuk said there are about 3,000 cells per dish, I will round up to 10,000 so there are extra oligos for multiple experiments 10^4 cells x 10^4 rolonies/cell = 10^8 rolonies in dish <br> <!-- not necessary b/c Hosuk only wants for RCA: 10^8 rolonies / 4,000 genes = 2.5 x 10^4 rolonies/gene (targets) <br> --> If we assume 50% of targets in reaction can assist circularization we need 2*10^8 targets = 3.32*10^-16 mol <br> At 100:1 that means we need 3.32*10^-14 mol of probes = 2.56*10^-18 mol of probe sets <br> Therefore the reaction set up below should be sufficient ====100:1 Probe:Target Calculations==== {| {{table}} | Probe set size||12964||probes |- | gDNA template||300||ng |- | gDNA MW||1.95E+12||g/mol (3x10^9 bp x 650 Da/bp + 157.9 Da) |- | template||1.5385E-19||mol |- | probe sets (100:1)||1.5385E-17||mol |- | probe MW (12,964 probes, 193nt)||814190000||g/mol (12,964 probes x (193 nt/probe x 325 Da/nt + 79 Da)) |- | amount probe required||1.25263E-08||g |- | ||12.5||ng |- | |} 16 x 20ul reactions: {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''16X Volume''' |- | gDNA (87ng/ul)||3.45||55.2 |- | Probe||0.745||11.92 |- | H2O||13.805||220.88 |- | 10X Ligase Buffer||2||32 |- | Total||20||320 |- | |} *40ul of Mineral oil on top *Ran thermacylcer program "Kun -> CpG"
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