Jie:LabNotes/CpgSeq/2009-8-7

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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;