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AlanFung:LabNotes/Capturing/2012-5-15
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==Sample List== {| {{table}} | ChIPK36me3||GM12878 |- | ChIPK36me3||P10 |- | ChIPK36me3||DF2 |- | ChIPK36me3||H0 |- | ChIPK36me3||H7 |- | ChIPK36me3 ||H14 |- | ChIPK36me3||H16 |- | || |- | ChIPK27me3||GM12878 |- | ChIPK27me3||P10 |- | ChIPK27me3||DF2 |- | ChIPK27me3||H0 |- | ChIPK27me3||H7 |- | ChIPK27me3 ||H14 |- | ChIPK27me3||H16 |- | || |- | WCE||GM12878 |- | WCE||P10 |- | WCE||DF2 |- | WCE||H0 |- | WCE||H7 |- | WCE||H14 |- | WCE||H16 |- | || |- | GM||cDNA |- | P10E3||cDNA |- | DF2||cDNA |- | H0||cDNA |- | H7||cDNA |- | H14||cDNA |- | H16||cDNA |- | || |- | GM||gDNA |- | P10E3||gDNA |- | DF2||gDNA |- | H0||gDNA |- | H7||gDNA |- | H14||gDNA |- | H16||gDNA |- | GmgDNA:H0gDNA::1:1 ||38 |} ==Sample== *H7 and H14 gDNA has the most amount and I would like to use these two samples for a first test {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''User ID''' | align="center" style="background:#f0f0f0;"|'''Date''' | align="center" style="background:#f0f0f0;"|'''Time''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''A260''' | align="center" style="background:#f0f0f0;"|'''A280''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''260/230''' | align="center" style="background:#f0f0f0;"|'''Constant''' | align="center" style="background:#f0f0f0;"|'''Cursor Pos.''' | align="center" style="background:#f0f0f0;"|'''Cursor abs.''' | align="center" style="background:#f0f0f0;"|'''340 raw''' | align="center" style="background:#f0f0f0;"|'''Vol''' | align="center" style="background:#f0f0f0;"|'''Total''' | align="center" style="background:#f0f0f0;"|'''to get 1000ng''' | align="center" style="background:#f0f0f0;"|'''Leftover for me''' |- | H7 gDNA||Default||4/5/2012||3:57 PM||184.33||3.687||1.787||2.06||2.23||50||230||1.652||0.001||100||18433||5.43||94.6 |- | H14 gDNA||Default||4/5/2012||3:58 PM||167.18||3.344||1.669||2||2.17||50||230||1.541||0.016||100||16718||5.98||94.0 |} ==Probes Needed== *Amount of probes *From the gel quantification we have 29.47ng/ul x 80ul =2.36ug of probes *Probe:target ratio: 200:1 H7 184.33ng/ul * 1.09ul = 200ng H14 167.18ng/ul * 1.20ul = 200ng To capture the 200ng DNA *Total probe molecule *200ng/3pgx200x100,000 *Mol of probe= 200ng/3pg*200*100,000/(6*10^23) *Concentration of probe set is 29.47 ng/ul. The length of the probe is approximately 100 base. ==Calculate probe concentration in nM== *Since 1base = 330Da (for single stranded DNA, 660Da for ds), *the concentration of probe is *29.47ng/ul*1x10^6ul/L*bp mol/330g*1/100bp = 893nM *the volume the probe needed is 200ng/3pg x 200 x 100,000 / {(6x10^23)x 893 nM} = 2.49 ul ==Capture System Setup== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H7 gDNA||1.09||H14 gDNA||1.20 |- | 10X Ampligase Buffer||4||10X Ampligase Buffer||4 |- | CES36K||2.49||CES36K||2.49 |- | H2O||12.42||H2O||12.31 |- | Total||20||Total||20 |} 95C 2min -> -0.02C/sec to 60C -> 60C 24h -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul Exo I&III -> 37C 2h -> 90C 5min -> 4C hold
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