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==breast cancer patient peripheral blood DNA sample capture by cpg97k== ===gDNA extraction from blood=== I used the Qiagen FlexiGene DNA kit to exact the DNA from blood. yield: 05192A18: 83.5ng/ul x 100ul; 05192B09: 72.7ng/ul x 100ul ===Bisulfite conversion of patient DNA === {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|'''No''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''sample volumn''' | align="center" style="background:#f0f0f0;"|'''ddH2O ''' | align="center" style="background:#f0f0f0;"|'''conversion reagents''' | align="center" style="background:#f0f0f0;"|'''conversed DNA concentration and volumn''' |- | ||05192A18||83.5ng/ul x 1 tube||20ul||0ul||130ul||148.2ng/ul x 10ul |- | ||05192B09||72.7ng/ulx 1 tubes||20ul||0ul||130ul||127.8ng/ul x 10ul |- |} ===cpature by cpg97k === {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|'''No ''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | align="center" style="background:#f0f0f0;"|'''template+cpg97k_A(60ng/ul_08/10) vol+suppress oligo+H2O''' | align="center" style="background:#f0f0f0;"|'''template+cpg97k_B(60ng/ul_08/10) vol+suppressor oligo+H2O''' |- |||05192A18 ||148.2ng/ul ||1ul||3+1.5ul+1ul+3.5ul||3+1.5ul+1ul+3.5ul |- |||05192B09 ||127.8ng/ul ||1ul||3+1.5ul+1ul+3.5ul||3+1.5ul+1ul+3.5ul |- |} PCR Template 15ul x4 2X iProof Mastermix 50ul AmpF6.3SoL (10uM) 4ul AmpR6.3SoL (10uM) 4ul 50X SYBG I 0.4ul H2O 26.6ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold. Qiaquick purification and e-gel size selection. ===PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40=== I did the dUTP_PCR with template from the Qiaquick purified captured targets of 97k. For each targets, I did 200ul PCR reaction. reaction system x8 H2O 42.6ul 340.8ul 2x Master mix 50ul 400ul dUTP(1mM) 2ul 16ul AmpF6.3NH2(10uM) 2ul 16ul AmpR6.3NH2(10uM) 2ul 16ul 50x SYBG I 0.4ul 3.2ul template 0.25ul/each for e-gel purified cpg97k Total 100ul 1400ul Purify with qiaquick column. Quantify with nanodrop and mix them with 1:1 ratio. ===USER and S1 digestion=== add 3ul USER to 30ul of each samples. 37C for 1h. 10 x S1 nuclease buffer: 4 ul DNA after USER digestion: 33ul S1 nuclease (10U/ul): 1ul ddH2O 2ul 37C 10mins. Minelute cloumn purify. Elute in 18ul H2O. ===endrepair with enzymatic end-repair kit=== x3 17 ul DNA 2.5 ul 10X End-Repair Buffer 7.5 2.5 ul dNTP Mix 7.5 3 ul End-Repair Enzyme Mix 9 25 ul Total reaction volume Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O. === A tail addition=== x3 Blunt-ended DNA 10ul 10 each 10X Klenow buffer 1.6ul 4.8 1mM dATP 3ul 9 Klenow fragment (exo-) 1ul 3 37C 30min, purified with MinElute columns, eluted with 12ul EB. ===adaptor ligation=== x4 DNA 10ul 2x QuickLigase buffer (enzymatic) 15ul 60 20uM Adaptor oligo mix 3ul 12 T4 DNA QuickLigase (enzymatic) 2ul 8 Incubate at RT for 15 minutes. Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O. ===PCR=== x4 Template 10ul 2x iProof mix 50ul 200 Solexa_PCR_up (10uM) 4ul 16 Solexa_PCR_lo_PE (10uM) 4ul 16 H2O 37ul 148 50X SYBG I 0.2ul 0.8 98C 30sec -> 4 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 8 cycles of (98C 10sec -> 72C 15 sec)-> 72C 3min -> 15C hold. [[Image:20090817_breast_cancer cell shotgun lib.jpg|300px]]20090817_breast cancer cell shotgun lib I did the e-gel size selection and quantification with Q-PCR ===quantification of PCR amplicons=== I dilute the PhiX lib(10nM) to 1nM, 0.1nM, 0.025nM, 0.005nM. I dilute the sample lib with 1:10 and 1:50 ratio. Syb_FP5: ATGATACGGCGACCACCGAG Syb_RP7: CAAGCAGAAGACGGCATACGAG x 16 Template 1ul 2x iProof mix 25ul 350 syb_RP7 (100uM) 0.2ul Syb_FP5 (100uM) 0.2ul H2O 24ul 50X SYBG I 0.2ul 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold. [[Media:20090817_quantification of breast cancer WBC.xls|20090817_quantification of breast cancer WBC]] 05192A18: 8.63nM; 05192B09: 6.67nM;
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