Jie:LabNotes/CpgSeq/2009-8-7: Difference between revisions
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>Sam Chiang No edit summary |
>Sam Chiang No edit summary |
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(12 intermediate revisions by the same user not shown) | |||
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98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold. | 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 | Qiaquick purification and e-gel size selection. | ||
===PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40=== | ===PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40=== | ||
I did the dUTP_PCR with template from | 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 | |||
reaction | Purify with qiaquick column. Quantify with nanodrop and mix them with 1:1 ratio. | ||
2x | ===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; |
Latest revision as of 01:20, 18 August 2009
breast cancer patient peripheral blood DNA sample capture by cpg97k[edit]
gDNA extraction from blood[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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[edit]
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.
20090817_quantification of breast cancer WBC
05192A18: 8.63nM; 05192B09: 6.67nM;