Jie:LabNotes/CpgSeq/2009-8-7

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Revision as of 01:13, 18 August 2009 by >Sam Chiang
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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.