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= BSPP LMS cluster capture of colon cancer primary tumor DNA = [[noi:DMR220k_LabNotes|'''Calendar''']]<br> [[Noi/NOTES/2014-8-21| Probe prep]]<br> == Experimental plan == * Use MONOD V5 set A to capture DNA from colon tumor DNA * Test sensitivity of BSPP capture of this probe set on 10, 20, 100 and 200ng == Bisulfite conversion and ssDNA quantification == * I did bisulfite conversion of 5 colon cancer DNA sample in duplicates using EZ DNA Methylation-Lightning™ Kit * I followed the instruction provided by manufacturer {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume for 600ng (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''H2O to 20ul''' |- | 6-T-1||43.40||13.82||6.18 |- | 6-T-2||31.60||18.99||1.01 |- | 6-T-3||31.50||19.05||0.95 |- | 6-T-4||41.70||14.39||5.61 |- | 6-T-5||30.50||19.67||0.33 |} 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. Continue to desulfonation :- Elute with 18ul Elution Buffer :- The volume left after Elution ~16ul :- Quantify concentration and recovery yield by Qubit ssDNA assay kit {| {{table}} | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:80px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|''' Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 16ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''% Recovery''' |- | 6-T-1_1||136||ng/mL||1||200||27.20||435.20||72.53 |- | 6-T-1_2||144||ng/mL||1||200||28.90||462.40||77.07 |- | 6-T-2_1||92.2||ng/mL||1||200||18.40||294.40||49.07 |- | 6-T-2_2||95||ng/mL||1||200||19.00||304.00||50.67 |- | 6-T-3_1||137||ng/mL||1||200||27.40||438.40||73.07 |- | 6-T-3_2||131||ng/mL||1||200||26.30||420.80||70.13 |- | 6-T-4_1||117||ng/mL||1||200||23.40||374.40||62.40 |- | 6-T-4_2||134||ng/mL||1||200||26.80||428.80||71.47 |- | 6-T-5_1||133||ng/mL||1||200||26.60||425.60||70.93 |- | 6-T-5_2||140||ng/mL||1||200||27.90||446.40||74.40 |} == BSPP capture set up == ==== Probe calculation ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="width:100px;background:#f0f0f0;"|'''200 : 1''' | align="center" style="background:#f0f0f0;"|'''Unit, calculation''' |- | Probe size||73,574|| |- | Template||10||ng |- | Human gDNA MW||1.950E+12||g/mol (3E+09 * 650Da/bp+157.9Da) |- | Human gDNA (10ng)||5.128E-21||mol (10ng/1.950E+12 g/mol) |- | Probe required (200:1)||1.026E-18||mol (2.5641E-20 * 200) |- | Probe MW (73,574 probes, 110nt)||2.636E+09||g/mol (73,574 probes * (110nt * 325Da/nt + 79Da)) |- | Amount probe required||2.704E-09||g |- | Amount probe required||2.7||ng |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Input bis-DNA (ng)''' | align="center" style="background:#f0f0f0;"|'''MONOD V5 set A amount (ng)''' |- | 10||2.70 |- | 20||5.40 |- | 100||27.00 |- | 200||54.00 |}
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