Jie:LabNotes/CpgSeq/2009-8-7
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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
No | sample | sample concentration | sample volumn | ddH2O | conversion reagents | 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
No | sample | sample concentration | 10xLigase buffer | template+cpg97k_A(60ng/ul_08/10) vol+suppress oligo+H2O | 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.
File:20090817 breast cancer cell shotgun lib.jpg20090817_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.